Pharmaceutical composition for subcutaneous injection comprising human hyaluronidase ph20 variant and drug

ABSTRACT

The present disclosure relates to a pharmaceutical composition including (a) a drug and (b) a human PH20 variant. 
     The human PH20 variant included in the pharmaceutical composition according to the present disclosure includes amino acid residue substitution(s) in one or more regions selected from an alpha-helix 8 region (S347 to C381) and a linker region (A333 to R346) between alpha-helix 7 and alpha-helix 8 in wild-type human PH20 having the amino acid sequence of SEQ ID NO: 1, wherein amino acid residue(s) located at the N-terminus or the C-terminus is(are) selectively cleaved. In addition, the pharmaceutical composition according to the present disclosure may further include a pharmaceutically acceptable additive, particularly a stabilizer. 
     The pharmaceutical composition according to the present disclosure can maximize the therapeutic effect of a drug used in combination therewith, due to the effect of human PH20 variants.

TECHNICAL FIELD

The present disclosure relates to a pharmaceutical composition includinga human hyaluronidase PH20 variant having enhanced enzymatic activityand thermal stability and one or more drugs, and a method of treating adisease using the same.

The pharmaceutical composition according to the present disclosure maypreferably be used for subcutaneous injection.

BACKGROUND ART

Drugs which should be administered in a high dose or in multiple doses,especially antibody drugs and the like, are generally administered viaintravenous injection, and such injection takes about 90 minutes orlonger, an additional preparation procedure should be accompanied forintravenous injection, thus both a patient, doctors and medical staffare inconvenienced, and additional costs are incurred. In contrast,subcutaneous injection has the advantage of enabling immediateadministration, but the absorption rate is relatively low compared tointravenous injection, and when the injection amount is 3-5 mL or more,it may cause swelling and pain at the injection site, as absorptionoccurs slowly. As For this reason, subcutaneous injection of proteintherapeutic agents is usually limited to solution injection of a smallamount of 2 mL or less. However, upon subcutaneous administration (orsubcutaneous injection) of hyaluronidase along with a therapeutic drug,hyaluronic acid distributed in the extracellular matrix is hydrolyzed bythe action of hyaluronidase, and thus the viscosity of the subcutaneousarea is reduced and the permeability of a substance is increased, andtherefore, a high dose or multiple doses of a medicine can easily bedelivered into the body.

There are six types of hyaluronidase genes in humans: Hya11, Hya12,Hya13, Hya14, HyalPS1, and PH20/SPAM1 Hya11 and Hya12 are expressed inmost tissues, and PH20/SPAM1 (hereinafter, referred to as PH20) isexpressed in the sperm cell membrane and the acrosomal membrane. HyalPS1is not expressed because it is a pseudogene. PH20 is an enzyme (EC3.2.1.35) that cleaves β-1,4 bonds between N-acetylglucosamine andglucuronic acid, which are sugars constituting hyaluronic acid. Humanhyaluronidase PH20 has an optimal pH of 5.5, but exhibits some activityeven at a pH of 7-8, whereas other human hyaluronidases, includingHya11, have an optimal pH of 3-4 and have very weak activity at a pH of7-8. The pH of subcutaneous areas in a human is about 7.4, which issubstantially neutral, and thus, among various types of hyaluronidases,PH20 is widely applied in clinical use. Examples of the clinical use ofPH20 include subcutaneous injection of an antibody therapeutic agent,use as an eye relaxant and an anesthetic additive in ophthalmic surgery,use in increase the access of an anticancer therapeutic agent to thetumor cells by hydrolyzing hyaluronic acid in the extracellular matrixof tumor cells, and use in promoting the resorption of body fluids andblood, which are excessively present in tissue.

Meanwhile, currently commercially available PH20 is in a form extractedfrom the testes of cattle or sheep. Examples thereof include Amphadase®(bovine hyaluronidase) and Vitrase® (sheep hyaluronidase).

Bovine testicular hyaluronidase (BTH) is obtained by removing a signalpeptide and 56 amino acids on the C-terminus from bovine wild-type PH20during post-translational modification. BTH is also a glycoprotein, andhas a mannose content of 5% and a glucosamine content of 2.2%, based onthe total constitution thereof including amino acids (Borders andRaftery, 1968). When animal-derived hyaluronidase is repeatedlyadministered to the human body at a high dose, a neutralizing antibodycan be produced, and other animal-derived biomaterials contained asimpurities in addition to PH20 may cause an allergic reaction. Inparticular, the use of PH20 extracted from cattle is limited due toconcern over mad cow disease. In order to overcome these problems,studies on recombinant human PH20 proteins have been conducted.

Recombinant human PH20 proteins have been reported to be expressed inyeast (P. pastoris), DS-2 insect cells, animal cells, and the like (Chenet al., 2016, Hofinger et al., 2007). The recombinant PH20 proteinsproduced in insect cells and yeast differ from human PH20 in terms ofthe pattern of N-glycosylation during post-translational modification.

Among hyaluronidases, the protein structures of Hya11 (PDB ID: 2PE4)(Chao et al., 2007) and bee venom hyaluronidase (PDB ID: 1FCQ, 1FCU,1FCV) have been identified. Hya11 is composed of two domains, acatalytic domain and an EGF-like domain, and the catalytic domain is inthe form of (β/α)₈, in which an alpha helix and a beta-strand, whichcharacterize the secondary structure of the protein, are each repeatedeight times (Chao et al., 2007). The EGF-like domain is completelyconserved in variants in which the C-terminus of Hya11 is spliceddifferently. The amino acid sequences of Hya11 and PH20 are 35.1%identical, and the protein tertiary structure of PH20 has not yet beenfound.

In a structural/functional relationship study of human PH20, theC-terminal region of PH20 was found to be important for proteinexpression and enzymatic activity, and in particular, it has beenreported that termination of the C-terminus with amino acids 477-483 isimportant for enzymatic expression and activity (Frost, 2007). Theactivity of full-length PH20 (amino acids 1-509) or a pH20 varianthaving a C-terminus truncated at position 467 was merely 10% or less ofthat of a pH20 variant having a C-terminus truncated at one site amongpositions 477 to 483 (Frost, 2007). Halozyme Therapeutics developedrHuPH20 (amino acids 36-482), which is a recombinant protein in whichthe C-terminus of mature PH20 was cleaved at Y482 (Bookbinder et al.,2006; Frost, 2007).

Meanwhile, although research is ongoing to develop various therapeuticdrugs in the form of subcutaneous injections using human PH20, theproblem of low stability of human PH20 itself still remains unsolved.

Against this technical background, the inventors of the presentdisclosure confirmed that human PH20 variants, including one or moreamino acid residue substitutions in an alpha-helix 8 region (S347 toC381) and a linker region (A333 to R346) between alpha-helix 7 andalpha-helix 8 in the amino acid sequence of wild-type hyaluronidasePH20, and in which some of amino acids located at the N-terminus and/orthe C-terminus of PH20 are cleaved, had very high enzymatic activity andthermal stability, and thus filed a patent application therefor (PCT/KR2019/009215).

The inventors of the present application also confirmed that the PH20variants according to the present disclosure may be applied topharmaceutical compositions or formulations including drugs, e.g.,antibody drugs, particularly high-dose anti-HER2 antibodies or immunecheckpoint antibodies, and accordingly, pharmaceutical compositions andformulations according to the present disclosure including PH20 variantsalong with drugs such as anti-HER2 antibodies or immune checkpointantibodies can be used for subcutaneous injection, and the activities ofdrugs such as antibody drugs and the PH20 variants are very stable andcan be maintained for a long time, thus completing the presentdisclosure.

DISCLOSURE Technical Problem

Therefore, the present disclosure has been made in view of the aboveproblems, and it is an object of the present disclosure to provide anovel pharmaceutical composition including a PH20 variant havingenhanced enzymatic activity and thermal stability and a drug, whereinthe thermal stability and activity of the drug and the PH20 variant canbe maintained for a long time, particularly a pharmaceutical compositionthat can be used for subcutaneous injection.

It is another object of the present disclosure to provide a method oftreating a disease including administering the pharmaceuticalcomposition according to the present disclosure to a subject in need oftreatment.

Technical Solution

In accordance with the present disclosure, the above and other objectscan be accomplished by the provision of a pharmaceutical compositionincluding (a) a drug and (b) a PH20 variant.

The PH20 variant included in the pharmaceutical composition according tothe present disclosure may include one or more amino acid residuesubstitutions selected from the group consisting of S343E, M345T, K349E,L353A, L354I, N356E, and I361T in wild-type human PH20 having an aminoacid sequence of SEQ ID NO: 1, and may further include amino acidresidue substitution(s) in one or more regions selected from analpha-helix 8 region (S347 to C381) and/or a linker region (A333 toR346) between alpha-helix 7 and alpha-helix 8, wherein some amino acidresidues located at an N-terminus and/or a C-terminus are selectivelycleaved.

The pharmaceutical composition according to the present disclosure mayfurther include one or more selected from pharmaceutically acceptableadditives, particularly a buffer, a stabilizer, and a surfactant.

The pharmaceutical composition according to the present disclosure maybe used in the form of an injection formulation for subcutaneousinjection.

DESCRIPTION OF DRAWINGS

The above and other objects, features and other advantages of thepresent disclosure will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIG. 1A illustrates size-exclusion chromatography chromatograms oftrastuzumab in a stability test under harsh conditions at 45° C., andFIG. 1B illustrates a change in the purity of a trastuzumab monomericprotein according to formulation in a stability test under harshconditions at 45° C.;

FIG. 2 illustrates the results of measuring the protein aggregationtemperatures of formulations including trastuzumab and a novel PH20variant HP46;

FIG. 3A is a weak cation exchange (WCX) chromatogram of trastuzumab in astability test under harsh conditions at 45° C., FIG. 3B illustrateschanges (%) in relative amounts of acidic variants in formulations in astability test under harsh conditions at 45° C., FIG. 3C illustrateschanges (%) in relative amounts of main peaks for formulations in astability test under harsh conditions at 45° C., and FIG. 3D illustrateschanges (%) in relative amounts of basic variants in formulations in astability test under harsh conditions at 45° C.;

FIG. 4 illustrates changes in the purity of a trastuzumab monomericprotein in formulations 5-7 in a stability test under harsh conditionsat 45° C.;

FIG. 5A illustrates changes (%) in relative amounts of acidic variantsin formulations 5-7 in a stability test under harsh conditions at 45°C., FIG. 5B illustrates changes (%) in relative amounts of main peaksaccording to formulations 5, 6, and 7 in a stability test under harshconditions at 45° C., and FIG. 5C illustrates changes (%) in relativeamounts of basic variants according to formulations 5, 6, and 7 in astability test under harsh conditions at 45° C.;

FIG. 6A illustrates the results of measuring the residual enzymaticactivity of a Herceptin subcutaneous injection formulation (HerceptinSC), trastuzumab+wild-type PH20 (HW2), and trastuzumab+PH20 variant HP46on day 0 and day 1 in a stability test under harsh conditions at 40° C.,and FIG. 6B illustrates the results of measuring the residual enzymaticactivity of the Herceptin subcutaneous injection formulation,trastuzumab+wild-type PH20 (HW2), and trastuzumab+PH20 variant HP46 onday 0 and day 1 in a stability test under harsh conditions at 45° C.;

FIG. 7 illustrates size-exclusion chromatography analysis results offormulations 8-10 in a stability test under harsh conditions at 40° C.for 14 days;

FIG. 8A illustrates the results of measuring changes in protein particlesize of formulations 8-10 using DLS equipment, and FIG. 8B illustratesthe results of measuring protein aggregation temperatures;

FIG. 9A illustrates a weak cation exchange (WCX) chromatogram offormulation 8 in a stability test under harsh conditions at 40° C., FIG.9B illustrates changes (%) in relative amounts of acidic variants informulations 8-10 in a stability test under harsh conditions at 40° C.,FIG. 9C illustrates changes (%) in relative amounts of main peaks forformulations 8-10 in a stability test under harsh conditions at 40° C.,and FIG. 9D illustrates changes (%) in relative amounts of basicvariants in formulations 8-10 in a stability test under harsh conditionsat 40° C.;

FIG. 10 illustrates changes (%) in relative enzymatic activity offormulations 8-10 in a stability test under harsh conditions at 40° C.;

FIG. 11 illustrates changes in the purity of trastuzumab monomers offormulations 11-13 in a stability test under harsh conditions at 40° C.;

FIG. 12A illustrates a weak cation exchange (WCX) chromatogram offormulation 11 in a stability test under harsh conditions at 40° C.,FIG. 12B illustrates changes (%) in relative amounts of acidic variantsin formulations 11-13 in a stability test under harsh conditions at 40°C., FIG. 12C illustrates changes (%) in relative amounts of main peaksfor formulations 11-13 in a stability test under harsh conditions at 40°C., and FIG. 12D illustrates changes (%) in relative amounts of basicvariants in formulations 11-13 in a stability test under harshconditions at 40° C.;

FIG. 13 illustrates changes (%) in relative enzymatic activity offormulations 11-13 in a stability test under harsh conditions at 40° C.;

FIG. 14 illustrates changes in the purity of rituximab monomers offormulations 14-16 in a stability test under harsh conditions at 40° C.;

FIG. 15 illustrates changes in relative enzymatic activity offormulations 14-16 in a stability test under harsh conditions at 40° C.;

FIG. 16 illustrates changes in relative enzymatic activity offormulations 17 and 18 in a stability test under harsh conditions at 40°C.;

FIG. 17 illustrates size-exclusion chromatography analysis results offormulations 19-22 at 40° C.;

FIG. 18 illustrates changes in relative enzymatic activity offormulations 19-22 in a stability test under harsh conditions at 40° C.;

FIG. 19 illustrates changes in enzymatic activity according to changesin pH for recombinant human PH20 and HP46; and

FIG. 20 illustrates experimental results of pharmacokinetics of aHerceptin subcutaneous injection product (Herceptin SC) and a Herceptinsubcutaneous injection biosimilar candidate (trastuzumab+HP46; HerceptinSC BS) in 9-week-old Sprague-Dawley rats, wherein Herceptin and theHerceptin biosimilar candidate were injected at 18 mg/kg each, and thesubcutaneous injection formulation contained 100 units of rHuPH20 and100 units of HP46 (at pH 5.3).

DETAILED DESCRIPTION AND EXEMPLARY EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as those generally understood by one of ordinaryskill in the art to which the invention pertains. In general, thenomenclature used herein is well known and commonly used in the art.

An embodiment of the present disclosure relates to a pharmaceuticalcomposition including (a) a drug and (b) a PH20 variant, and thepharmaceutical composition according to the present disclosure may beused for the prevention or treatment of a disease, and is preferablyused for subcutaneous injection.

The human PH20 variant included in the pharmaceutical compositionaccording to the present disclosure has some amino acid residuesubstitutions in the region corresponding to an alpha-helix regionand/or a linker region thereof, preferably an alpha-helix 8 region (S347to C381) and/or a linker region (A333 to R346) between alpha-helix 7 andalpha-helix 8, more preferably an amino acid region among T341 to N363,and most preferably T341 to I361, L342 to I361, S343 to I361, I344 toI361, M345 to I361, or M345 to N363, in the amino acid sequence ofwild-type PH20 (having the amino acid sequence of SEQ ID NO: 1),preferably mature wild-type PH20 (having the sequence consisting of L36to 5490 in the amino acid sequence of SEQ ID NO: 1).

In the present disclosure, “mature wild-type PH20” refers to a proteincomprising amino acid residues L36 to S490 of SEQ ID NO: 1, which lackM1 to T35, which form a signal peptide, and A491 to L509, which are notrelated to the substantial function of PH20, in the amino acid sequenceof wild-type PH20 having the sequence of SEQ ID NO: 1.

TABLE 1  Amino acid sequence of wild-type PH20 (SEQ ID NO: 1)MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLTLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKEYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSATMEIVSILFLIISSVASL

Specifically, the PH20 variant or fragment thereof included in thepharmaceutical composition according to the present disclosure includesone or more mutations, preferably amino acid residue substitutionsselected from the group consisting of S343E, M345T, K349E, L353A, L354I,N356E, and I361T, and most preferably one or more amino acid residuesubstitutions selected from the group consisting of L354I and N356E, inwild-type PH20 having the sequence of SEQ ID NO: 1.

In the present disclosure, the term “PH20 variant” is intended toinclude mutation of some amino acid residues, preferably substitution ofamino acid residues in the sequence of wild-type human PH20, as well asthe deletion of some amino acid residues at the N-terminus and/or theC-terminus together with such substitution of amino acid residues, andis used with substantially the same meaning as the expression “PH20variant or a fragment thereof.”

The inventors of the present disclosure have verified novel PH20variants or fragments thereof with increased enzymatic activity andthermal stability compared to wild-type PH20 can be provided throughprevious studies, based on experimental results in which, enzymaticactivity and a protein aggregation temperature (Tagg) at a neutral pHare increased, when the amino acid sequences of an alpha-helix 8 regionand a linker region between alpha-helix 7 and alpha-helix 8 of humanPH20 are partially substituted with the amino acid sequences of analpha-helix 8 region and a linker region between alpha-helix 7 andalpha-helix 8 of Hya11 with high hydrophilicity.

Accordingly, the PH20 variant included in the pharmaceutical compositionaccording to the present disclosure includes one or more amino acidresidue substitutions selected from the group consisting of S343E,M345T, K349E, L353A, L354I, N356E, and I361T, preferably one or moreamino acid residue substitutions selected from the group consisting ofL354I and N356E, in the amino acid sequence of wild-type PH20 (havingthe amino acid sequence of SEQ ID NO: 1), preferably mature wild-typePH20 (having a sequence consisting of L36 to 5490 in the amino acidsequence of SEQ ID NO: 1),

in which one or more amino acid residues in the region corresponding toan alpha-helix region and/or a linker region thereof, preferably in analpha-helix 8 region (S347 to C381) and/or a linker region (A333 toR346) between alpha-helix 7 and alpha-helix 8, more preferably in anamino acid region corresponding to T341 to N363, T341 to I361, L342 toI361, S343 to I361, I344 to I361, M345 to I361, or M345 to N363, aresubstituted.

Particularly, in the PH20 variant included in the pharmaceuticalcomposition according to the present disclosure, the alpha-helix 8region (S347 to C381) and/or the linker region (A333 to R346) ofalpha-helix 7 and alpha-helix 8 of wild-type PH20, preferably maturewild-type PH20, may be substituted with some amino acid residues in theamino acid sequence of a corresponding region of Hya11 having thesequence of SEQ ID NO: 51 (see Tables 2 and 3), but the presentdisclosure is not limited thereto.

TABLE 2  Amino acid sequence of wild-type Hyal1 (SEQ ID NO: 51)MAAHLLPICALFLTLLDMAQGFRGPLLPNRPFTTVWNANTQWCLERHGVDVDVSVFDVVANPGQTFRGPDMTIFYSSQLGTYPYYTPTGEPVFGGLPQNASLIAHLARTFQDILAAIPAPDFSGLAVIDWEAWRPRWAFNWDTKDIYRQRSRALVQAQHPDWPAPQVEAVAQDQFQGAARAWMAGTLQLGRALRPRGLWGFYGFPDCYNYDFLSPNYTGQCPSGIRAQNDQLGWLWGQSRALYPSIYMPAVLEGTGKSQMYVQHRVAEAFRVAVAAGDPNLPVLPYVQIFYDTTNHFLPLDELEHSLGESAAQGAAGVVLWVSWENTRTKESCQAIKEYMDTTLGPFILNVTSGALLCSQALCSGHGRCVRRTSHPKALLLLNPASFSIQLTPGGGPLSLRGALSLEDQAQMAVEF KCRCYPGWQAPWCERKSMW

TABLE 3 Comparison between alpha helixes and amino acid sequences ofPH20 and Hyal1 Amino acid sequence of Amino acid sequence of Alpha helixPH20 Hyal1 Alpha-helix 1 P56 to D65 N39 to G48 Alpha-helix 3 S119 toM135 S101 to I117 Alpha-helix 4′ K161 to N176 K144 to H159 Alpha-helix 4S180 to R211 P163 to R194 Alpha-helix 5 F239 to S256 P222 to S239Alpha-helix 6 A274 to D293 K257 to G277 Alpha-helix 7 S317 to G332 P299to G314 Alpha-helix 8 S347 to C381 T329 to C363

More specifically, the PH20 variant or fragment thereof included in thepharmaceutical composition according to the present disclosurepreferably includes an amino acid residue substitution of L354I and/orN356E in the amino acid sequence of wild-type PH20, preferably maturewild-type PH20,

and preferably further includes an amino acid residue substitution atone or more positions selected from T341 to N363, particularly at one ormore positions selected from the group consisting of T341, L342, S343,I344, M345, S347, M348, K349, L352, L353, D355, E359, I361, and N363,but the present disclosure is not limited thereto, and

more preferably, further includes one or more amino acid residuesubstitutions selected from the group consisting of T341S, L342W, S343E,I344N, M345T, S347T, M348K, K349E, L352Q, L353A, D355K, E359D, I361T,and N363G, but the present disclosure is not limited thereto.

Preferably, the PH20 variant or fragment thereof included in thepharmaceutical composition according to the present disclosure mayinclude an amino acid residue substitution selected from M345T, S347T,M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T,

and may further include one or more amino acid residue substitutionsselected from the group consisting of T341S, L342W, S343E, I344N, andN363G, but the present disclosure is not limited thereto.

More preferably, the PH20 variant or fragment thereof included in thepharmaceutical composition according to the present disclosure mayinclude, but is not limited to, any one substitution selected from thefollowing groups:

(a) T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q,L353A, L354I, D355K, N356E, E359D, and I361T;

(b) L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A,L354I, D355K, N356E, E359D, and I361T;

(c) M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E,E359D, and I361T;

(d) M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E,E359D, I361T, and N363G;

(e) I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K,N356E, E359D, and I361T; and

(f) S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I,D355K, N356E, E359D, and I361T.

In the present disclosure, an expression, which is described byone-letter amino acid residue code together with numbers, such as“S347”, means the amino acid residue at the corresponding position inthe amino acid sequence of SEQ ID NO: 1.

For example, “S347” means that the amino acid residue at position 347 inthe amino acid sequence of SEQ ID NO: 1 is serine. In addition, “S347T”means that serine at position 347 of SEQ ID NO: 1 is substituted forthreonine.

The PH20 variant included in the pharmaceutical composition according tothe present disclosure is interpreted as including variants in which theamino acid residue at a specific amino acid residue position isconservatively substituted.

As used herein, the term “conservative substitution” refers tomodifications of a PH20 variant that involves the substitution of one ormore amino acids with amino acids having similar biochemical propertiesthat do not cause loss of the biological or biochemical function of thecorresponding PH20 variant.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined and are well known in the art. These families include aminoacids with basic side chains (e.g., lysine, arginine, and histidine),amino acids with acidic side chains (e.g., aspartic acid and glutamicacid), amino acids with uncharged polar side chains (e.g., glycine,asparagine, glutamine, serine, threonine, tyrosine, and cysteine), aminoacids with nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, and tryptophan), aminoacids with beta-branched side chains (e.g., threonine, valine, andisoleucine), and amino acids with aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, and histidine).

It is anticipated that the PH20 variant included in the pharmaceuticalcomposition according to the present disclosure will still retain theactivity thereof despite having conservative amino acid substitutions.

In addition, the PH20 variant or fragment thereof included in thepharmaceutical composition according to the present disclosure isinterpreted as including PH20 variants or fragments thereof havingsubstantially the same function and/or effect as those/that of the PH20variant or the fragment thereof according to the present disclosure, andhaving an amino acid sequence homology of at least 80% or 85%,preferably at least 90%, more preferably at least 95%, and mostpreferably at least 99% with the PH20 variant or fragment thereofaccording to the present disclosure.

The PH20 variants according to the present disclosure have increasedexpression levels in animal cells and an increased protein refoldingrate, thereby having increased thermal stability compared to maturewild-type PH20. Furthermore, the enzymatic activity of the PH20 variantsexceeded or was similar to that of mature wild-type PH20 despite theincrease in thermal stability.

Meanwhile, it is known that, when some amino acids at the C-terminus,such as 5490, of mature wild-type PH20 are additionally cleaved, theenzymatic activity is reduced, but the PH20 variants according to thepresent disclosure showed increased thermal stability and increased orsimilar enzymatic activities compared to mature wild-type PH20 eventhough the C-terminus of mature wild-type PH20 has an additionallycleaved sequence. In addition, the PH20 variants maintained enzymaticactivities thereof when up to five amino acid residues were cleaved fromthe N-terminal amino acids, which indicates that residues starting fromP41 of the N-terminus played an important role in protein expression andenzymatic activity.

Accordingly, the PH20 variant included in the pharmaceutical compositionaccording to the present disclosure includes some amino acid residuesubstitutions in the alpha-helix 8 region (S347 to C381) and/or thelinker region (A333 to R346) between alpha-helix 7 and alpha-helix 8 ofwild-type PH20, and further includes some amino acid residue deletionsat the C-terminus and/or the N-terminus, but the present disclosure isnot limited thereto.

In one embodiment, the PH20 variant included in the pharmaceuticalcomposition according to the present disclosure may include some aminoacid residue deletions at the N-terminus resulting from cleavage beforean amino acid residue selected from the group consisting of M1 to P42 atthe N-terminus of the amino acid sequence of SEQ ID NO: 1, preferablybefore an amino acid residue L36, N37, F38, R39, A40, P41, or P42,and/or some amino acid residue deletions at the C-terminus resultingfrom cleavage after an amino acid residue selected from the groupconsisting of V455 to W509 at the C-terminus, preferably after an aminoacid residue selected from the group consisting of V455 to 5490, andmost preferably, after an amino acid reside V455, C458, D461, C464,1465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478,1480, Y482, A484, P486, T488, or 5490.

The expression “cleavage before L36, N37, F38, R39, A40, P41, or P42 atthe N-terminus” means, respectively, that all amino acid residues fromM1 to T35 immediately before L36, all amino acid residues from M1 to L36immediately before N37, all amino acid residues from M1 to N37immediately before F38, all amino acid residues from M1 to F38immediately before R39, all amino acid residues from M1 to R39immediately before A40, all amino acid residues from M1 to A40immediately before P41, or all amino acid residues from M1 to P41immediately before P42 in the amino acid sequence of SEQ ID NO: 1 arecleaved and removed. The expression “cleavage before M1 at theN-terminus of SEQ ID NO: 1” means that no cleavage occurs at theN-terminus.

In addition, the expression “cleavage after V455, C458, D461, C464,1465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478,1480, Y482, A484, P486, T488, or 5490 of the C-terminus” means cleavageand removal of the amino acid residue following the V455, C458, D461,C464, 1465, D466, A467, F468, K470, P472, M473, E474, T475, E476, P478,1480, Y482, A484, P486, T488, or 5490, respectively, in the sequence ofSEQ ID NO: 1. For example, cleavage after 5490 means cleavage between5490 and A491.

Preferably, the human PH20 variant included in the pharmaceuticalcomposition according to the present disclosure may have an amino acidsequence selected from the group consisting of the amino acid sequencesof SEQ ID NOS: 5 to 50, more preferably the amino acid sequence of SEQID NO: 44, but the present disclosure is not limited thereto. In PH20variants constructed in specific embodiments according to the presentdisclosure, the sequences of substituted or cleaved amino acids areshown in Table 4 below.

TABLE 4  Amino acid sequences of PH20 variants accordingto the present disclosure and the substitution/cleavage properties thereof Seguence Name Number Substitution SequenceHM1 5 12 amino acids are  LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMsubstituted with SLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI M345T, S347T,TGVIVNGGIPQKISLQDHLDKAKKDITFYMPVDNL M348K, K349E,GMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQ L352Q, L353A,NVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLL L354I, D355K,RPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKR N356E, E359D,NDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRN I361T, and N363G.RVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKF LSQDELVYTFGETVALGASGIVIWGILSI T RTKE S C QAIKE YMDT T L G PYIINVILAAKMCSQVTCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGK PTLEDLEQFSEKEYCSCYSTLSCKEKADVKDIDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM2 6 7 amino acids LNFRAPPVIPNVPFLWAWNAPSEFCLGKEDEPLDM are substitutedSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI withTGVIVNGGIPQKISLQDHLDKAKKDITFYMPVDNL Y365F, I367L,GMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQ L371S, A372G,NVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLL K374L, M375L, andRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKR V379A.NDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRN RVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGILSITRTKES CQAIKEYMDTTLNP F I L NVI SG A LL CSQA LCQEQG VCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKEYCSCYSTLSCKEKADVKDIDAV DVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM3 7 19 amino acids are LNFRAPPVIPNVPFLWAWNAPSEFCLGKEDEPLDMsubstituted with SLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI M345T, S347T,TGVIVNGGIPQKISLQDHLDKAKKDITFYMPVDNL M348K, K349E,GMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQ L352Q, L353A,NVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLL L354I, D355K,RPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKR N356E, E359D,NDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRN I361T, N363G,RVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKF Y365F, I367L,LSQDELVYTEGETVALGASGIVIWGILSI T R T KE S L371S, A372G, C QAIKE YM D T TL G P F I L NVT SG A LL CSQ A LCQEQG K374L, M375L, andVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGK V379A.PTLEDLEQFSEKEYCSCYSTLSCKEKADVKDIDAV DVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM4 8 17 amino acids are LNFRAPPVIPNVPFLWAWNAPSEFCLGKEDEPLDMsubstituted with SLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI G340V, T341S,TGVIVNGGIPQKISLQDHLDKAKKDITFYMPVDNL L342W, S343E,GMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQ I344N, M345T,NVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLL S347T, M348K,RPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKR K349E, L352Q,NDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRN L353A, L354I,RVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKF D355K, N356E,LSQDELVYTEGETVALGASGIVIW VSWEN TRTKE S E359D, I361T, and C QAIKE YM D TT L G PYIINVTLAAKMCSQVLCQEQG N363G. VCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKEYCSCYSTLSCKEKADVKDIDAV DVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM6 9 11 amino acid LNFRAPPVIPNVPFLWAWNAPSEFCLGKEDEPLDM residues areSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI substituted withTGVIVNGGIPQKISLQDHLDKAKKDITFYMPVDNL M345T, S347T,GMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQ M348K, K349E,NVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLL L352Q, L353A,RPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKR L354I, D355K,NDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRN N356E, E359D, andRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKF I361T. LSQDELVYTEGETVALGASGIVIWGILSITRTKE S C QAIKE YM D T T LNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGK PTLEDLEQFSEKEYCSCYSTLSCKEKADVKDIDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM7 10 16 amino acids are LNFRAPPVIPNVPFLWAWNAPSEFCLGKEDEPLDM substituted withSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI G340V, T3415 L342W,TGVIVNGGIPQKISLQDHLDKAKKDITFYMPVDNL 5343E, I344N,GMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQ M345T, S347T,NVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLL M348K, K349E,RPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKR L352Q, L353A,NDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRN L354I, D355K,RVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKF N356E, E359D, andLSQDELVYTEGETVALGASGIVIW VSWENT R TKE S I361T. C QAIKE YM D T TLNPYIINVTLAAKMCSQVLCQEQG VCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKEYCSCYSTLSCKEKADVKDIDAV DVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM8 11 12 amino acids are LNFRAPPVIPNVPFLWAWNAPSEFCLGKEDEPLDMsubstituted with SLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI I344N, M345T,TGVIVNGGIPQKISLQDHLDKAKKDITFYMPVDNL S347T, M348K,GMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQ K349E, L352Q,NVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLL L353A, L354I,RPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKR D355K, N356E,NDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRN E359D, and I361T.RVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKF LSQDELVYTEGETVALGASGIVIWGILS NT RTKE S C QAIKE YM D T T LNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGK PTLEDLEQFSEKEYCSCYSTLSCKEKADVKDIDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM9 12 13 amino acids areLNFRAPPVIPNVPFLWAWNAPSEFCLGKEDEPLDM substituted withSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI S343E, I344N,TGVIVNGGIPQKISLQDHLDKAKKDITFYMPVDNL M345T, S347T,GMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQ M348K, K349E,NVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLL L352Q, L353A,RPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKR L354I, D355K,NDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRN N356E, E359D, andRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKF I361T. LSQDELVYTEGETVALGASGIVIWGILENT R TKE S C QAIKE YM D T T LNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGK PTLEDLEQFSEKEYCSCYSTLSCKEKADVKDIDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM10 13 14 amino acidLNFRAPPVIPNVPFLWAWNAPSEFCLGKEDEPLDM residues areSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI substituted withTGVIVNGGIPQKISLQDHLDKAKKDITFYMPVDNL L342W, S343E,GMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQ I344N, M345T,NVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLL S347T, M348K,RPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKR K349E, L352Q,NDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRN L353A, L354I,RVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKF D355K, N356E,LSQDELVYTEGETVALGASGIVIWGT WENT R TKE S E359D, and 1361T. C QAIKE YM D TT LNPYIINVTLAAKMCSQVLCQEQG VCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKEYCSCYSTLSCKEKADVKDIDAV DVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM11 14 13 amino acid LNFRAPPVIPNVPFLWAWNAPSEFCLGKEDEPLDM residues areSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI substituted withTGVIVNGGIPQKISLQDHLDKAKKDITFYMPVDNL M345T, S347T,GMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQ M348K, K349E,NVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLL L352Q, L353A,RPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKR L354I, D355K,NDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRN N356E, E359D,RVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKF I361T, Y365F, andLSQDELVYTFGETVALGASGIVIWGTLSI T R TKE S I367L. C QAIKE YM D T T LNP F IL NVTLAAKMCSQVLCQEQG VCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAV DVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM12 15 15 amino acid LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDM residues areSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI substituted withTGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNL M345T, S347T,GMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQ M348K, K349E,NVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLL L352Q, L353A,RPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKR L354I, D355K,NDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRN N356E, E359D,RVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKF I361T, Y365F,LSQDELVYTFGETVALGASGIVIWGTLSI T R TKE S I367L, L371S, and C QAIKE YM D TT LNP F I L NVTSGAKMCSQVLCQEQG A372G.VCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGK PTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM13 16 11 amino acidFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSL residues areFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITG substituted withVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGM M345T, S347T,AVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNV M348K, K349E ,QLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRP L352Q, L353A,NHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRND L354I, D355K,DLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRV N356E, E359D, andREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLS I361T, and cleavageQDELVYTFGETVALGASGIVIWGTLSI T R TKE SC Q is performed before AIKE YM D TT LNPYIINVTLAAKMCSQVTCQEQGVC residue F38 at theIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPT N-terminus.LEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDV CIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM14 17 11 amino acids are LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMsubstituted with SLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI M345T, S347T,TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNL M348K, K349E,GMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQ L352Q, L353A,NVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLL L354I, D355K,RPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKR N356E, E359D, andNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRN I361T, and cleavageRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKF is performed afterLSQDELVYTFGETVALGASGIVIWGTLSI T R TKE S the carboxyl group C QAIKE YM DT T LNPYIINVTLAAKMCSQVLCQEQG of I465.VCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGK PTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCI HM15 18 11 amino acids areLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDM substituted withSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI M345T, S347T,TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNL M348K, K349E,GMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQ L352Q, L353A,NVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLL L354I, D355K,RPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKR N356E, E359D, andNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRN I361T, and cleavageRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKF is performed afterLSQDELVYTFGETVALGASGIVIWGTLSI T R TKE S the carboxyl group C QAIKE YM DT T LNPYIINVTLAAKMCSQVLCQEQG of F468.VCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGK PTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF HM16 19 11 amino acids areLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDM substituted withSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI M345T, S347T,TGVIVNGGIPQKISLQDHLDKAKKDITFYMPVDNL M348K, K349E,GMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQ L352Q, L353A,NVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLL L354I, D355K,RPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKR N356E, E359D, andNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRN I361T, and cleavageRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKF is performed afterLSQDELVYTFGETVALGASGIVIWGILSI T R TKE S the carboxyl group C QAIKE YM DT T LNPYIINVTLAAKMCSQVLCQEQG of P471.VCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGK PTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKP HM17 20 Amino acids L36 to FRGPLLPNRPFLWAWNAPSEFCLGKFDEPLDMSLF V47 are substitutedSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGV with FRGPLLPNR, andTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMA 11 amino acids areVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQ substituted withLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPN M345T, S347T,HLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDD M348K, K349E, LSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVR L352Q, L353A, EAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQ L354I, D355K, DELVYTFGETVALGASGIVIWGILSI T R TKE SC QA N356E, E359D, and IKE YM D T TLNPYIINVTLAAKMCSQVLCQEQGVCI I361T. RKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPILEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVC IADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM18 21 Amino acids L36 to FRGPLLPNRPFTTV WNAPSEFCLGKFDEPLDMSLFA52 are substituted SFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGV withTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMA FRGPLLPNRPFTTV, andVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQ 11 amino acids areLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPN substituted withHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDD M345T, S347T,LSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVR M348K, K349EEAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQ L352Q, L353ADELVYTFGETVALGASGIVIWGILSI T R TKE SC QA L354I D355K IKE YM D T TLNPYIINVTLAAKMCSQVLCQEQGVCI N356E, E359D, andRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPIL I361T.EDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVC IADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM19 22 14 amino acid FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSL residues areFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITG substituted withVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGM L342W, S343E,AVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNV I344N, M345T,QLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRP S347T, M348K,NHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRND K349E, L352Q,DLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRV L353A, L354I,REAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLS D355K, N356E,QDELVYTFGETVALGASGIVIWGT WENT R TKE SC Q E359D, and I361T, AIKE YM D T TLNPYIINVTLAAKMCSQVLCQEQGVC and cleavage isIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPT performed beforeLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDV residue F38 at the CIADGVCIDAFLKN-terminus and after residue K470 at the C-terminus. HM20 2314 amino acid FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSL residues are FSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITG substituted withVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGM L342W, S343E,AVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNV I344N, M345T,QLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRP S347T, M348K,NHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRND K349E, L352Q,DLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRV L353A, L354I,REAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLS D355K, N356E,QDELVYTEGETVALGASGIVIWGT WENT R TKE SC Q E359D, and I361T, AIKE YM D T TLNPYIINVTLAAKMCSQVLCQEQGVC and cleavage isIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPT performed beforeLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDV residue F38 at the CIADGVCIDAFN-terminus and after residue F468 at the C-terminus. HM21 2415 amino acid LNFRAPPVIPNVPFLWAWNAPSEFCLGKEDEPLDM residues areSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI substituted withTGVIVNGGIPQKISLQDHLDKAKKDITFYMPVDNL T341S, L342W,GMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQ S343E, I344N,NVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLL M345T, S347T,RPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKR M348K, K349E,NDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRN L352Q, L353A,RVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKF L354I, D355K,LSQDELVYTEGETVALGASGIVIWG SWENT R TKE S N356E, E359D, and C QAIKE YM D TT LNPYIINVTLAAKMCSQVLCQEQG I361T. VCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKEYCSCYSTLSCKEKADVKDIDAV DVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM24 25 11 amino acid APPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFS residues areFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVT substituted withVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAV M345T, S347T IDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQL M348K, K349E,SLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNH L352Q, L353A,LWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDL L354I, D355K,SWLWNESTALYPSIYLNTQQSPVAATLYVRNRVRE N356E, E359D, andAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQD I361T, and cleavageELVYTEGETVALGASGIVIWGILSI T R TKE SC QAI is performed before KE YM D T TLNPYIINVILAAKMCSQVTCQEQGVCIR residue A40 at theKNWNTSTYLHLNPDNFAIQLEKGGKFTVRGKPTLE N-terminus.DLEQFSEKEYCSCYSTLSCKEKADVKDIDAVDVCI ADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM2526 11 amino acids are PVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIsubstituted with GSPRINATGQGVTIFYVDRLGYYPYIDSITGVIVN M345T, S347T,GGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVID M348K, K349E,WEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSL L352Q, L353A,TEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLW L354I, D355K,GYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSW N356E, E359D, andLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAI I361T, and cleavageRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDEL is performed beforeVYTEGETVALGASGIVIWGILSI T R TKE SC QAIKE residue P42 at the YM D T TLNPYIINVTLAAKMCSQVLCQEQGVCIRKN N-terminus.WNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDL EQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLS HM29 27 14 amino acidLNFRAPPVIPNVPFLWAWNAPSEFCLGKEDEPLDM residues areSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI substituted withTGVIVNGGIPQKISLQDHLDKAKKDITFYMPVDNL L342W, S343E,GMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQ I344N, M345T,NVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLL S347T, M348K,RPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKR K349E, L352Q,NDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRN L353A, L354I,RVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKF D355K, N356E,LSQDELVYTEGETVALGASGIVIWGT WENT R TKE S E359D, and I361T, C QAIKE YM D TT LNPYIINVTLAAKMCSQVLCQEQG and cleavage isVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGK performed beforePTLEDLEQFSEKEYCSCYSTLSCKEKADVKDIDAV residue L36 at the DVCIADGVCIDAN-terminus and after residue A467 at the C-terminus. HM30 2814 amino acid LNFRAPPVIPNVPFLWAWNAPSEFCLGKEDEPLDM resides areSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI substituted withTGVIVNGGIPQKISLQDHLDKAKKDITFYMPVDNL L342W, S343E,GMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQ I344N, M345T,NVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLL S347T, M348K,RPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKR K349E, L352Q,NDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRN L353A, L354I,RVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKF D355K, N356E,LSQDELVYTEGETVALGASGIVIWGT WENT R TKE S E359D, and I361T, C QAIKE YM D TT LNPYIINVTLAAKMCSQVLCQEQG and cleavage isVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGK performed beforePTLEDLEQFSEKEYCSCYSTLSCKEKADVKDIDAV residue L36 at the DVCIADGVCN-terminal and after residue C464 at the C-terminus. HM31 2914 amino acid LNFRAPPVIPNVPFLWAWNAPSEFCLGKEDEPLDM residues areSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI substituted withTGVIVNGGIPQKISLQDHLDKAKKDITFYMPVDNL L342W, S343E,GMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQ I344N, M345T,NVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLL S347T, M348K,RPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKR K349E, L352Q,NDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRN L353A, L354I,RVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKF D355K, N356E,LSQDELVYTEGETVALGASGIVIWGT WENT R TKE S E359D, and I361T, C QAIKE YM D TT LNPYIINVTLAAKMCSQVLCQEQG and cleavage isVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGK performed beforePTLEDLEQFSEKEYCSCYSTLSCKEKADVKDIDAV residue L36 at the DVCIADN-terminus and after residue D461 at the C-terminus. HM32 3014 amino acid LNFRAPPVIPNVPFLWAWNAPSEFCLGKEDEPLDM residues areSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI substituted withTGVIVNGGIPQKISLQDHLDKAKKDITFYMPVDNL L342W, S343E,GMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQ I344N, M345T,NVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLL S347T, M348K,RPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKR K349E, L352Q,NDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRN L353A, L354I,RVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKF D355K, N356E,LSQDELVYTEGETVALGASGIVIWGT WENT R TKE S E359D, and I361T, C QAIKE YM D TT LNPYIINVTLAAKMCSQVLCQEQG and cleavage isVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGK performed beforePTLEDLEQFSEKEYCSCYSTLSCKEKADVKDIDAV residue L36 at the DVCN-terminus and after residue C458 at the C-terminus. HM33 3114 amino acid LNFRAPPVIPNVPFLWAWNAPSEFCLGKEDEPLDM residues areSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI substituted withTGVIVNGGIPQKISLQDHLDKAKKDITFYMPVDNL L342W, S343E,GMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQ I344N, M345T,NVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLL S347T, M348K,RPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKR K349E, L352Q,NDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRN L353A, L354I,RVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKF D355K, N356E,LSQDELVYTEGETVALGASGIVIWGT WENT R TKE S E359D, and I361T, C QAIKE YM D TT LNPYIINVTLAAKMCSQVLCQEQG and cleavage isVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGK performed beforePTLEDLEQFSEKEYCSCYSTLSCKEKADVKDIDAV residue L36 at the N-terminus andafter residue V455 at the C-terminus. HP34 32 15 amino acidFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSL residues areFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITG substituted withVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGM T341S, L342W,AVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNV S343E, I344N,QLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRP M345T, S347T,NHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRND M348K, K349E,DLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRV L352Q, L353A,REAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLS L354I, D355K,QDELVYTEGETVALGASGIVIWGS WENT R TKE SC Q N356E, E359D, and AIKE YM D T TLNPYIINVTLAAKMCSQVLCQEQGVC I361T, and cleavageIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPT is performed beforeLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDV residue F38 at the CIADGVCIDAFLKN-terminus and after residue K470 at the C-terminus. HM35 3314 amino acid FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSL residues areFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITG substituted withVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGM L342W, S343E,AVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNV I344N, M345T,QLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRP S347T, M348K,NHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRND K349E, L352Q,DLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRV L353A, L354I,REAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLS D355K, N356E,QDELVYTEGETVALGASGIVIWGT WENT R TKE SC Q E359D, and I361T, AIKE YM D T TLNPYIINVTLAAKMCSQVLCQEQGVC and cleavage isIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPT performed beforeLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDV residue F38 at the CIADGVCIDAFLKPPN-terminus and after residue P472 at the C-terminus. HM36 3414 amino acid FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSL residues areFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITG substituted withVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGM L342W, S343E,AVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNV I344N, M345T,QLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRP S347T, M348K,NHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRND K349E, L352Q,DLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRV L353A, L354I,REAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLS D355K, N356E,QDELVYTEGETVALGASGIVIWGT WENT R TKE SC Q E359D, and I361T, AIKE YM D T TLNPYIINVTLAAKMCSQVLCQEQGVC and cleavage isIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPT performed beforeLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDV residue F38 at the CIADGVCIDAFLKPPMN-terminus and after residue M473 at the C-terminus. HM37 3514 amino acid FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSL residues areFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITG substituted withVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGM L342W, S343E,AVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNV I344N, M345T,QLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRP S347T, M348K,NHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRND K349E, L352Q,DLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRV L353A, L354I,REAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLS D355K, N356E,QDELVYTEGETVALGASGIVIWGT WENT R TKE SC Q E359D, and I361T, AIKE YM D T TLNPYIINVTLAAKMCSQVLCQEQGVC and cleavage isIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPT performed beforeLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDV residue F38 at the CIADGVCIDAFLKPPMEN-terminus and after residue E474 at the C-terminus. HM38 3614 amino acid FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSL residues are FSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITG substituted withVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGM L342W, S343E,AVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNV I344N, M345T,QLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRP S347T, M348K,NHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRND K349E, L352Q,DLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRV L353A, L354I,REAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLS D355K, N356E,QDELVYTEGETVALGASGIVIWGT WENT R TKE SC Q E359D, and I361T, AIKE YM D T TLNPYIINVTLAAKMCSQVLCQEQGVC and cleavage isIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPT performed beforeLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDV residue F38 at theCIADGVCIDAFLKPPMET N-terminus and after residue T475 at the C-terminus.HM39 37 14 amino acid FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSL residues areFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITG substituted withVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGM L342W, S343E,AVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNV I344N, M345T,QLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRP S347T, M348K,NHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRND K349E, L352Q,DLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRV L353A, L354I,REAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLS D355K, N356E,QDELVYTEGETVALGASGIVIWGT WENT R TKE SC Q E359D, and I361T, AIKE YM D T TLNPYIINVTLAAKMCSQVLCQEQGVC and cleavage isIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPT performed beforeLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDV residue F38 at theCIADGVCIDAFLKPPMETE N-terminus and after residue E476 at the C-terminus.HM40 38 11 amino acid NFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMS residues areLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSIT substituted withGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLG M345T, S347T,MAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQN M348K, K349E,VQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLR L352Q, L353A,PNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRN L354I, D355K,DDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNR N356E, E359D, andVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFL I361T, and cleavageSQDELVYTEGETVALGASGIVIWGILSI T R TKE SC is performed before QAIKE YM D TT LNPYIINVILAAKMCSQVTCQEQGV residue N37 at theCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKP N-terminus.TLEDLEQFSEKEYCSCYSTLSCKEKADVKDIDAVD VCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM41 39 11 amino acid RAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLF residues areSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGV substituted withTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMA M345T, S347T,VIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQ M348K, K349E,LSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPN L352Q, L353A,HLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDD L354I, D355K,LSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVR N356E, E359D, andEAIRVSKIPDAKSPLPVFAYTRIVETDQVLKFLSQ I361T, and cleavageDELVYTEGETVALGASGIVIWGILSI T R TKE SC QA is performed before IKE YM D TT LNPYIINVTLAAKMCSQVTCQEQGVCI residue R39 at theRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPIL N-terminus.EDLEQFSEKEYCSCYSTLSCKEKADVKDIDAVDVC IADGVCIDAFLKPPMETEEPQIFYNASPSTLSHM42 40 11 amino acid PPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSF residues areIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTV substituted withNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVI M345T, S347T,DWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLS M348K, K349E,LTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHL L352Q, L353A,WGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLS L354I, D355K,WLWNESTALYPSIYLNTQQSPVAATLYVRNRVREA N356E, E359D, andIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDE I361T, and cleavageLVYTEGETVALGASGIVIWGILSI T R TKE SC QAIK is performed before E YM D T TLNPYIINVTLAAKMCSQVTCQEQGVCIRK residue P41 at theNWNTSTYLHLNPDNFAIQLEKGGKFTVRGKPTLED N-terminus.LEQFSEKEYCSCYSTLSCKEKADVKDIDAVDVCIA DGVCIDAFLKPPMETEEPQIFYNASPSTLS HM4341 14 amino acid FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSL residues areFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITG substituted withVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGM L342W, S343E,AVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNV I344N, M345T,QLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRP S347T, M348K,NHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRND K349E, L352Q,DLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRV L353A, L354I,REAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLS D355K, N356E,QDELVYTEGETVALGASGIVIWGS WENT R TKE SC Q E359D, and I361T, AIKE YM D T TLNPYIINVTLAAKMCSQVLCQEQGVC and cleavage isIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPT performed beforeLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDV residue F38 at the CIADGVCIN-terminus and after residue 1465 at the C-terminus. HM44 4214 amino acid FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSL residues areFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITG substituted withVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGM L342W, S343E,AVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNV I344N, M345T,QLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRP S347T, M348K,NHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRND K349E, L352Q,DLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRV L353A, L354I,REAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLS D355K, N356E,QDELVYTEGETVALGASGIVIWGS WENT R TKE SC Q E359D, and I361T, AIKE YM D T TLNPYIINVTLAAKMCSQVLCQEQGVC and cleavage isIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPT performed beforeLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDV residue F38 at the CIADGVCIDN-terminus and after residue D466 at the C-terminus. HM45 4314 amino acid FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSL residues areFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITG substituted withVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGM L342W, S343E,AVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNV I344N, M345T,QLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRP S347T, M348K,NHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRND K349E, L352Q,DLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRV L353A, L354I,REAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLS D355K, N356E,QDELVYTEGETVALGASGIVIWGS WENT R TKE SC Q E359D, and I361T, AIKE YM D T TLNPYIINVTLAAKMCSQVLCQEQGVC and cleavage isIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPT performed beforeLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDV residue F38 at the CIADGVCIDAN-terminus and after residue A467 at the C-terminus. HP46 4415 amino acid FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSL residues areFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITG substituted withVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGM T341S, L342W,AVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNV S343E, I344N,QLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRP M345T, S347T,NHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRND M348K, K349E,DLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRV L352Q, L353A,REAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLS L354I, D355K,QDELVYTEGETVALGASGIVIWGS WENT R TKE SC Q N356E, E359D, and AIKE YM D T TLNPYIINVTLAAKMCSQVLCQEQGVC I361T, and cleavageIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPT is performed beforeLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDV residue F38 at the CIADGVCIDAFN-terminus and after residue F468 at the C-terminus. HM47 4514 amino acid FRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSL residues areFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITG substituted withVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGM L342W, S343E,AVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNV I344N, M345T,QLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRP S347T, M348K,NHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRND K349E, L352Q,DLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRV L353A, L354I,REAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLS D355K, N356E,QDELVYTEGETVALGASGIVIWGT WENT R TKE SC Q E359D, and I361T, AIKE YM D T TLNPYIINVTLAAKMCSQVLCQEQGVC and cleavage isIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPT performed beforeLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDV residue F38 at theCIADGVCIDAFLKPPMETEEP N-terminus and after residue P478at the C-terminus. HM48 46 14 amino acidFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSL residues areFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITG substituted withVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGM L342W, S343E,AVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNV I344N, M345T,QLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRP S347T, M348K,NHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRND K349E, L352Q,DLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRV L353A, L354I,REAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLS D355K, N356E,QDELVYTEGETVALGASGIVIWGT WENT R TKE SC Q E359D, and I361T, AIKE YM D T TLNPYIINVTLAAKMCSQVLCQEQGVC and cleavage isIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPT performed beforeLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDV residue F38 at theCIADGVCIDAFLKPPMETEEPQI N-terminal and after residue I480at the C-terminus. HM49 47 14 amino acidFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSL residues areFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITG substituted withVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGM L342W, S343E,AVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNV I344N, M345T,QLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRP S347T, M348K,NHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRND K349E, L352Q,DLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRV L353A, L354I,REAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLS D355K, N356E,QDELVYTEGETVALGASGIVIWGT WENT R TKE SC Q E359D, and I361T, AIKE YM D T TLNPYIINVTLAAKMCSQVLCQEQGVC and cleavage isIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPT performed beforeLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDV residue F38 at theCIADGVCIDAFLKPPMETEEPQIFY N-terminus and after residue Y482at the C-terminus. HM50 48 14 amino acidFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSL residues areFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITG substituted withVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGM L342W, S343E,AVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNV I344N, M345T,QLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRP S347T, M348K,NHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRND K349E, L352Q,DLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRV L353A, L354I,REAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLS D355K, N356E,QDELVYTEGETVALGASGIVIWGT WENT R TKE SC Q E359D, and I361T, AIKE YM D T TLNPYIINVTLAAKMCSQVLCQEQGVC and cleavage isIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPT performed beforeLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDV residue F38 at theCIADGVCIDAFLKPPMETEEPQIFYNA N-terminus and after residue A484at the C-terminus. HM51 49 14 amino acidFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSL residues areFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITG substituted withVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGM L342W, S343E,AVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNV I344N, M345T,QLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRP S347T, M348K,NHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRND K349E, L352Q,DLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRV L353A, L354I,REAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLS D355K, N356E,QDELVYTEGETVALGASGIVIWGT WENT R TKE SC Q E359D, and I361T, AIKE YM D T TLNPYIINVTLAAKMCSQVLCQEQGVC and cleavage isIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPT performed beforeLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDV residue F38 at theCIADGVCIDAFLKPPMETEEPQIFYNASP N-terminus and after residue P486at the C-terminus. HM52 50 14 amino acidFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSL residues areFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITG substituted withVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGM L342W, S343E,AVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNV I344N, M345T,QLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRP S347T, M348K,NHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRND K349E, L352Q,DLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRV L353A, L354I,REAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLS D355K, N356E,QDELVYTEGETVALGASGIVIWGT WENT R TKE SC Q E359D, and I361T, AIKE YM D T TLNPYIINVTLAAKMCSQVLCQEQGVC and cleavage isIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPT performed beforeLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDV residue F38 at theCIADGVCIDAFLKPPMETEEPQIFYNASPST N-terminus and after residue T488at the C-terminus.

Meanwhile, previous studies reported that the enzymatic activity ofwild-type PH20 changes depending on the cleavage positions of amino acidresidues located at the C-terminus. In the present disclosure, however,a specific alpha helix forming the secondary structure of PH20 wassubstituted with the alpha helix of other human hyaluronidase, therebyconstructing PH20 variants having higher stability than wild-type PH20,and in these variants, the interaction between the substitutedalpha-helix domain and other secondary structures of PH20 shows apattern different from that of wild-type PH20, so that the variants havea certain level of enzymatic activity or higher, regardless of thecleavage position at the C-terminus.

In addition, in the present disclosure, attempts were made to increasethe expression of a recombinant PH20 protein by using the signal peptideof other proteins exhibiting high protein expression levels in animalcells, instead of the original signal peptide of human PH20.

Therefore, in another embodiment, the PH20 variant included in thepharmaceutical composition according to the present disclosure mayinclude, at the N-terminus thereof, a signal peptide derived from humanhyaluronidase-1 (Hya11), a human growth hormone, or human serum albumin,instead of a signal peptide of wild-type PH20, which consists of M1 toT35, and preferably may include, as shown in Table 5, ahuman-growth-hormone-derived signal peptide having the amino acidsequence of MATGSRTSLLLAFGLLCLPWLQEGSA according to SEQ ID NO: 2, ahuman serum albumin-derived signal peptide having the amino acidsequence of MKWVTFISLLFLFSSAYS according to SEQ ID NO: 3, or a humanHya11-derived signal peptide having the amino acid sequence ofMAAHLLPICALFLTLLDMAQG according to SEQ ID NO: 4, but the presentdisclosure is not limited thereto.

TABLE 5  Amino acid sequence of signal peptide of humangrowth hormone, human serum albumin, or human Hyal1 Origin of signal SEQpeptide Amino acid sequence ID NO: Human growth MATGSRTSLLLAFGLLCLPWLQEGSA 2 hormone Human serum  MKWVTFISLLFLFSSAYS 3albumin Human Hyal1 MAAHLLPICALFLTLLDMAQG 4

Among the PH20 variants included in the pharmaceutical compositionaccording to the present disclosure, a variant having a 6×His-tagattached to the C-terminus was named HM, and a variant without the6×His-tag was named HP. In addition, mature wild-type PH20 (L36-5490)with a 6×His-tag attached to the C-terminus thereof was named WT, andmature wild-type PH20 (L36 to Y482) without the 6×His-tag and in whichthe C-terminus is cleaved after Y482 was named HW2.

HP46 (SEQ ID NO: 44) is a human PH20 variant obtained by modeling aprotein structure using Hya11 (PDB ID: 2PE4) (Chao et al., 2007), whichis human hyaluronidase, with a known protein tertiary structure, andthen substituting the amino acid sequence of amino acids of alpha-helix8 and the linker region between alpha-helix 7 and alpha-helix 8 with theamino acid sequence of Hya11, and subjecting the N-terminal to cleavageat F38 and subjecting the C-terminus to cleavage after F468. Inparticular, alpha-helix 8 is located outside the protein tertiarystructure of PH20 and has less interaction with neighboring alphahelices or beta-strands than other alpha helices. In general, enzymaticactivity and thermal stability have a trade-off relationshiptherebetween, and thus the higher the thermal stability of a protein,the lower the enzymatic activity, whereas, when the enzymatic activityis increased due to an improvement in the flexibility of the proteinstructure, the thermal stability tends to be reduced. However, thespecific activity of HP46, measured by Turbidimetric assay at a pH of7.0, was about 46 units/μg, which was evaluated to be about two timesthat of wild-type PH20, which was about 23 units/μg.

The thermal stability of a protein may be evaluated based on a meltingtemperature Tm, at which 50% of the protein tertiary structure isdenatured, and on an aggregation temperature Tagg, at which aggregationbetween proteins occurs. In general, the aggregation temperature of aprotein tends to be lower than the melting temperature thereof. Thealpha-helix 8 of Hya11 exhibits greater hydrophilicity than thealpha-helix 8 of PH20. The substituted alpha-helix 8 of Hya11 increasesthe protein surface hydrophilicity of HP46, thereby causing the effectof delaying aggregation between proteins that occurs due to hydrophobicinteractions, and thus the aggregation temperature is 51° C., which isobserved to be an increase of 4.5° C. compared to the aggregationtemperature of wild-type PH20, which is 46.5° C.

HP46 is a variant in which amino acid residues in the alpha-helix 8 andthe linker region between alpha-helix 7 and alpha-helix 8 aresubstituted, wherein T341 is substituted with serine. When amino acidresidue 341 is threonine, the enzyme activity is similar to that ofwild-type PH20, but upon substitution with serine, the enzyme activityis increased about 2-fold, and it was confirmed that, even in asubstrate gel assay, the resultant variant hydrolyzed hyaluronic acid 5to 6 times more than wild-type PH20. Substrate gel assay involvesprotein denaturation and refolding processes, which means that theprotein tertiary structure refolding and restoration of HP46 areenhanced compared to wild-type PH20.

The amount of the PH20 variant in the pharmaceutical compositionaccording to the present disclosure is at least 50 units/mL, preferablyin the range of 100 units/mL to 20,000 units/mL, more preferably in therange of about 150 units/mL to about 18,000 units/mL, still morepreferably in the range of 1,000 units/mL to 16,000 units/mL, and mostpreferably in the range of 1,500 units/mL to 12,000 units/mL.

Examples of the drug included in the pharmaceutical compositionaccording to the present disclosure include, but are not limited to,protein drugs, antibody drugs, small molecules, aptamers, RNAi,antisenses, and cellular therapeutic agents such as chimeric antigenreceptor (CAR)-T or CAR-natural killer (NK), and it is possible to usenot only currently commercially available drugs but also drugs inclinical trials or under development.

As the drug, a protein drug or an antibody drug may preferably be used.

The “protein drug” included in the pharmaceutical composition accordingto the present disclosure is a drug that consists of amino acids, andthus exhibits the effect of treating or preventing a disease through theactivity of a protein, is a drug consisting of a protein other than theantibody drug, and may be selected from the group consisting of acytokine, a therapeutic enzyme, a hormone, a soluble receptor and afusion protein thereof, insulin or an analogue thereof, bonemorphogenetic protein (BMP), erythropoietin, and a serum-derivedprotein, but the present disclosure is not limited thereto.

The cytokine included in the pharmaceutical composition according to thepresent disclosure may be selected from the group consisting ofinterferon, interleukin, colony-stimulating factor (CSF), tumor necrosisfactor (TNF), and tissue growth factor (TGF), but the present disclosureis not limited thereto.

The therapeutic enzyme may include, but is not limited to,β-glucocerebrosidase and agalsidase β.

The soluble receptor included in the pharmaceutical compositionaccording to the present disclosure is an extracellular domain of thereceptor, and the fusion protein thereof is a protein in which the Fcregion or the like of an antibody is fused to the soluble receptor. Thesoluble receptor is a soluble form of a receptor to which adisease-related ligand binds, and examples thereof include a form inwhich an Fc region is fused to the TNF-α soluble receptor (e.g., aproduct containing the ingredient etanercept and forms similar thereto),a form in which an Fc region is fused to the VEGF soluble receptor (aproduct containing the ingredient alefacept and forms similar thereto),a form in which an Fc region is fused to CTLA-4 (e.g., a productcontaining the ingredient abatacept or belatacept and forms similarthereto), a form in which an Fc region is fused to the interleukin 1soluble receptor (e.g., a product containing the ingredient rilonaceptand forms similar thereto), and a form in which an Fc region is fused tothe LFA3 soluble receptor (e.g., a product containing the ingredientalefacept and forms similar thereto), but the present disclosure is notlimited thereto.

The hormone included in the pharmaceutical composition according to thepresent disclosure refers to a hormone injected into the body or ananalog thereof for the treatment or prevention of diseases caused byhormone deficiency and the like, and examples thereof include, but arenot limited to, human growth hormone, estrogen, and progesterone.

The serum-derived protein included in the pharmaceutical compositionaccording to the present disclosure is a protein present in plasma, andincludes both proteins extracted from plasma and produced recombinantproteins, and examples thereof may include, but are not limited to,fibrinogen, von Willebrand factor, albumin, thrombin, factor II (FII),factor V (FV), factor VII (FVII), factor IX (FIX), factor X (FX), andfactor XI (FXI).

The antibody drug included in the pharmaceutical composition accordingto the present disclosure may be a monoclonal antibody drug or apolyclonal antibody drug.

The monoclonal antibody drug according to the present disclosure is aprotein containing a monoclonal antibody and a monoclonal antibodyfragment that are capable of specifically binding to an antigen relatedto a specific disease. The monoclonal antibody also includes abispecific antibody, and the protein containing a monoclonal antibody orfragment thereof conceptually includes an antibody-drug conjugate (ADC).

Examples of the antigen related to a specific disease include 4-1BB,integrin, amyloid beta, angiopoietin (angiopoietin 1 or 2), angiopoietinanalog 3, B-cell-activating factor (BAFF), B7-H3, complement 5, CCR4,CD3, CD4, CD6, CD11a, CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD62,CD79b, CD80, CGRP, Claudin-18, complement factor D, CTLA4, DLL3, EGFreceptor, hemophilia factor, Fc receptor, FGF23, folate receptor, GD2,GM-CSF, HER2, HER3, interferon receptor, interferon gamma, IgE, IGF-1receptor, interleukin 1, interleukin 2 receptor, interleukin 4 receptor,interleukin 5, interleukin 5 receptor, interleukin 6, interleukin 6receptor, interleukin 7, interleukin 12/23, interleukin 13, interleukin17A, interleukin 17 receptor A, interleukin 31 receptor, interleukin 36receptor, LAG3, LFA3, NGF, PVSK9, PD-1, PD-L1, RANK-L, SLAMF7, tissuefactor, TNF, VEGF, VEGF receptor, and von Willebrand factor (vWF), butthe present disclosure is not limited thereto.

The followings are, but are not limited to, proteins includingmonoclonal antibodies or monoclonal antibody fragments against theantigens related to a specific disease:

utomilumab as an anti-4-1BB antibody;

natalizumab, etrolizumab, vedolizumab, and bimagrumab as antibodiesagainst integrin;

bapineuzumab, crenezumab, solanezumab, aducanumab, and gantenerumab asantibodies against amyloid beta;

antibodies against angiopoietin such as AMG780 against angiopoietin 1and 2, MEDI 3617 and nesvacumab against angiopoietin 2, and vanucizumabwhich is a bispecific antibody against angiopoietin 2 and VEGF;

evinacumab as an antibody against angiopoietin analog 3;

tabalumab, lanalumab, and belimumab as antibodies againstB-cell-activating factor (BAFF);

omburtamab as an antibody against B7-H3;

ravulizumab and eculizumab as antibodies against complement 5;

mogamulizumab as an antibody against CCR4;

otelixizumab, teplizumab, and muromonab as antibodies against CD3,tebentafusp as a bispecific antibody against GP100 and CD3, blinatumomabas a bispecific antibody against CD19 and CD3, and REGN1979 as abispecific antibody against CD20 and CD3;

ibalizumab and zanolimumab as antibodies against CD4;

itolizumab as an antibody against CD6;

efalizumab as an antibody against CD11a;

inebilizumab, tafasitamab, and loncastuximab tesirine which is an ADC,as antibodies against CD19;

ocrelizumab, ublituximab, obinutuzumab, ofatumumab, rituximab,tositumomab, and ibritumomab tiuxetan which is an ADC, as antibodiesagainst CD20;

epratuzumab, inotuzumab ozogamicin which is an ADC, and moxetumomabpasudotox as antibodies against CD22;

brentuximab vedotin as an ADC against CD30;

vadastuximab talirine and gemtuzumab ozogamicin as ADCs against CD33;

daratumumab and isatuximab as antibodies against CD38;

alemtuzumab as an antibody against CD52;

crizanlizumab as an antibody against CD62;

polaruzumab vedotin as an ADC against CD79b;

galiximab as an antibody against CD80;

eptinezumab, fremanezumab, galcanezumab, and erenumab as antibodiesagainst CGRP;

zolbetuximab as an antibody against Claudin-18;

lampalizumab as an antibody against complement factor D;

tremelimumab, zalifrelimab, and ipilimumab as antibodies against CTLA4;

rovalpituzumab tesirine as an ADC against DLL3;

cetuximab, depatuxizumab, zalutumumab, necitumumab, and panitumumab asantibodies against the EGF receptor;

emicizumab as a bispecific antibody against coagulation factor IX andfactor X, which are hemophilia factors;

nipocalimab and rozanolixizumab as antibodies against the Fc receptor;

burosumab as an antibody against FGF23;

farletuzumab as an antibody against the folate receptor and mirvetuximabsoravtansine as an ADC against the folate receptor;

dinutuximab and naxitamab as antibodies against GD2;

otilimab as an antibody against GM-CSF;

margetuximab, pertuzumab, and trastuzumab as antibodies against HER2,and trastuzumab deruxtecan, trastuzumab emtansine, and trastuzumabduocarmazine as ADCs against HER2;

patritumab as an antibody against HER3;

anifrolumab as an antibody against interferon receptor;

emapalumab as an antibody against interferon gamma;

ligelizumab and omalizumab as antibodies against IgE;

dalotuzumab, figitumumab, and teprotumumab as antibodies against theIGF-1 receptor;

gebokizumab and canakinumab as antibodies against interleukin 1;

daclizumab and basiliximab as antibodies against the interleukin 2receptor;

dupilumab as an antibody against the interleukin 4 receptor;

mepolizumab and reslizumab as antibodies against interleukin 5;

benralizumab as an antibody against the interleukin 5 receptor;

clazakizumab, olokizumab, sirukumab, and siltuximab as antibodiesagainst interleukin 6;

sarilumab, satralizumab, tocilizumab, and REGN88 as antibodies againstthe interleukin 6 receptor;

secukinumab as an antibody against interleukin 7;

ustekinumab and briakinumab as antibodies against interleukin 12/23;

lebrikizumab and tralokinumab as antibodies against interleukin 13;

ixekizumab and bimekizumab as antibodies against interleukin 17A;

brodalumab as an antibody against interleukin 17 receptor A;

brazikumab, guselkumab, risankizumab, tildrakizumab, and mirikizumab asantibodies against interleukin 23;

nemolizumab as an antibody against the interleukin 31 receptor;

spesolimab as an antibody against the interleukin 36 receptor;

relatlimab as an antibody against LAG3;

narsoplimab as an antibody against NASP2;

fasinumab and tanezumab as antibodies against NGF;

alirocumab, evolocumab, and bococizumab as antibodies against PVSK9;

lambrolizumab, balstilimab, camrelizumab, cemiplimab, dostarlimab,prolgolimab, shintilimab, spartalizumab, tislelizumab, pembrolizumab,and nivolumab as antibodies against PD-1;

atezolizumab, avelumab, envafolimab, and durvalumab as antibodiesagainst PD-L1, and bintrafusp alpha as a bispecific antibody against TGFbeta and PD-L1;

denosumab as an antibody against RANK-L;

elotuzumab as an antibody against SLAMF7;

concizumab and marstacimab as antibodies against tissue factor;

antibodies against TNF, particularly TNFα, including infliximab,adalimumab, golimumab, the antibody fragment certolizumab pegol, andozoralizumab which is a bispecific antibody against TNF and albumin;

antibodies against VEGF, including brolucizumab, ranibizumab,bevacizumab, and faricimab which is a bispecific antibody against VEGFand Ang2;

ramucirumab as an antibody against the VEGF receptor; and

caplacizumab as an antibody against vWF.

Meanwhile, the overexpression of human epidermal growth factor receptor2 (HER2), which promotes cell division, is observed in about 20-25% ofbreast cancer patients, and HER2-over-expressed breast cancer progressesquickly, is aggressive, and has a low response to chemotherapy comparedto HER2-low-expressed breast cancer, and thus the prognosis thereof isunfavorable. Trastuzumab, which is a monoclonal antibody drug targetingHER2, specifically binds to HER2 on the surfaces of HER2-overexpressingcancer cells to inhibit the signal transduction of cell replication andproliferation, thereby slowing tumor progression. Trastuzumab wasapproved by the United States Food and Drug Administration (FDA) in 1998for the treatment of breast cancer in the United States, and in 2003 bythe Korea Food and Drug Administration (KFDA). Since then, the efficacyof trastuzumab was also recognized in HER2-overexpressing gastriccancer, and thus has been used as a therapeutic agent for gastriccancer.

A Roche's Herceptin intravenous injection formulation (commercial name:Herceptin) consists of 440 mg of trastuzumab as a main ingredient, andlyophilized trastuzumab is mixed with physiological saline and injectedinto a vein. On the other hand, a subcutaneous injection formulation oftrastuzumab (commercial name: Herceptin SC) is a 5 mL liquidformulation, and contains 600 mg (120 mg/mL) of trastuzumab as a mainingredient, and includes, as additives, 20 mM histidine (pH 5.5), 210 mMtrehalose, 10 mM methionine, 0.04% polysorbate 20, and 10,000 units ofrHuPH20 (2,000 Units/mL, 0.004%, 40 μg/mL).

The shelf life of Herceptin subcutaneous injection formulations is 21months. The intravenous injection formulation of trastuzumab is in alyophilized form and has a shelf life of 30 months, but the subcutaneousinjection formulation of trastuzumab is in a liquid state and has ashort shelf life of 21 months. For this reason, it can be estimated thatthe stability of one or more of trastuzumab and recombinant humanhyaluronidase PH20 in liquid formulations is limited.

In this context, in the present disclosure, in view of thecharacteristics of the PH20 variant according to the present disclosure,in which, compared to wild-type human hyaluronidase PH20 and recombinanthuman PH20 available from Halozyme, the PH20 variant not only hasincreased enzymatic activity, but also has a high measured proteinaggregation temperature, thus exhibiting enhanced thermal stability, theshelf life of the subcutaneous injection formulation is set to along-term period, preferably 21 months or longer. The content of theantibody drug in the pharmaceutical composition according to the presentdisclosure may be in the range of 5 mg/mL to 500 mg/mL, preferably 20mg/mL to 200 mg/mL, more preferably 100 mg/mL to 150 mg/mL, and mostpreferably 120±18 mg/mL, for example, about 110 mg/mL, about 120 mg/mL,or about 130 mg/mL.

The polyclonal antibody included in the pharmaceutical compositionaccording to the present disclosure is preferably a serum antibodyextracted from serum such as immune globulin, but is not limitedthereto.

In the case of a small-molecule compound, any drug that requires a rapideffect for prevention or treatment may be used without limitation. Forexample, morphine-based painkillers may be used (Thomas et al., 2009).In addition, when used as a therapeutic agent for tissue necrosis causedby anticancer drugs, the small-molecule compound may be used alone or incombination with antidote drugs such as Vinca alkaloids and Taxanes(Kreidieh et al., 2016).

The pharmaceutical composition according to the present disclosure mayfurther include one or more selected from the group consisting of abuffer, a stabilizer, and a surfactant.

The buffer included in the composition according to the presentdisclosure may be used without limitation, as long as it enablesrealization of a pH of 4 to 8, preferably 5 to 7, and the buffer ispreferably one or more selected from the group consisting of malate,formate, citrate, acetate, propionate, pyridine, piperazine, cacodylate,succinate, 2-(N-morpholino)ethanesulfonic acid (MES), histidine, Tris,bis-Tris, phosphate, ethanolamine, carbonate,piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), imidazole, BIS-TRISpropane, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),3-(N-morpholino) propanesulfonic acid) (MOPS), hydroxyethyl piperazineethane sulfonic acid (HEPES), pyrophosphate, and triethanolamine, morepreferably a histidine buffer, e.g., L-histidine/HCl, but is not limitedthereto.

The concentration of the buffer may be in the range of 0.001 mM to 200mM, preferably 1 mM to 50 mM, more preferably 5 mM to 40 mM, and mostpreferably 10 mM to 30 mM.

Stabilizers in the composition according to the present disclosure maybe used without limitation, as long as they are commonly used in the artfor the purpose of stabilizing proteins, and preferably, the stabilizersmay be, for example, one or more selected from the group consisting ofcarbohydrates, sugars or hydrates thereof, sugar alcohols or hydratesthereof, and amino acids.

Carbohydrates, sugars, or sugar alcohols used as the stabilizer may beone or more selected from the group consisting of trehalose or hydratesthereof, sucrose, saccharin, glycerol, erythritol, threitol, xylitol,arabitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol,inositol, volemitol, isomalt, maltitol, polyglycitol, cyclodextrin,hydroxylpropyl cyclodextrin, and glucose, but is not limited thereto.

The amino acid may be one or more selected from the group consisting ofglutamine, glutamic acid, glycine, lysine, lysilysine, leucine,methionine, valine, serine, selenomethionine, citrulline, arginine,asparagine, aspartic acid, ornithine, isoleucine, taurine, theanine,threonine, tryptophan, tyrosine, phenylalanine, proline, pyrrolysine,histidine, and alanine, but is not limited thereto.

The concentration of the sugars or sugar alcohols used as a stabilizerin the pharmaceutical composition according to the present disclosuremay be in the range of 0.001 mM to 500 mM, preferably 100 mM to 300 mM,more preferably 150 mM to 250 mM, and most preferably 180 mM to 230 mM,and particularly, may be about 210 mM.

In addition, the concentration of amino acids used as a stabilizer inthe pharmaceutical composition according to the present disclosure maybe in the range of 1 mM to 100 mM, preferably 3 mM to 30 mM, morepreferably 5 mM to 25 mM, and most preferably 7 mM to 20 mM, andspecifically, may be in the range of 8 mM to 15 mM.

The composition according to the invention may further include asurfactant.

Preferably, the surfactant may be a non-ionic surfactant such aspolyoxyethylene-sorbitan fatty acid ester (polysorbate or Tween),polyethylene-polypropylene glycol, polyoxyethylene-stearate,polyoxyethylene alkyl ethers, e.g., polyoxyethylene monolauryl ether,alkylphenyl polyoxyethylene ether [Triton-X], and apolyoxyethylene-polyoxypropylene copolymer [Poloxamer and Pluronic], andsodium dodecyl sulfate (SDS), but is not limited thereto.

More preferably, polysorbate may be used. The polysorbate may bepolysorbate 20 or polysorbate 80, but is not limited thereto.

The concentration of the nonionic surfactant in the pharmaceuticalcomposition according to the present disclosure may be in the range of0.0000001% (w/v) to 0.5% % (w/v), preferably 0.000001% (w/v) to 0.4%(w/v), more preferably 0.00001% (w/v) to 0.3% (w/v), and most preferably0.001% (w/v) to 0.2% (w/v).

In one embodiment, the pharmaceutical composition according to thepresent disclosure may include 50-350 mg/mL of an antibody, for example,an anti-HER2 antibody or an immune checkpoint antibody, histidine bufferproviding a pH of 5.5±2.0, 10-400 mM α,α-trehalose, 1-50 mM methionine,and 0.0000001% (w/v) to 0.5% (w/v) of polysorbate.

In a more specific embodiment, the pharmaceutical composition accordingto the present disclosure may include 120 mg/mL of an anti-HER2 antibodyor an immune checkpoint antibody, 20 mM histidine buffer that provides apH of 5.5±2.0, 210 mM α,α-trehalose, 10 mM methionine, and 2,000units/mL of a PH20 variant, and may further include 0.005% (w/v) to 0.1%(w/v) polysorbate.

The pharmaceutical composition according to the present disclosure maybe administered via intravenous injection, subcutaneous injection,intramuscular injection, intraperitoneal injection, endothelialadministration, topical administration, intranasal administration,intrapulmonary administration, intrarectal administration, and the like,and subcutaneous administration is preferably performed via subcutaneousinjection, and it is more preferable to use the pharmaceuticalcomposition as an injection formulation for subcutaneous injection.

Therefore, another embodiment of the present disclosure provides aformulation including the pharmaceutical composition according to thepresent disclosure, preferably an injection formulation for subcutaneousinjection.

The injection formulation for subcutaneous injection may be provided ina ready-to-inject form without an additional dilution process, and maybe provided after being contained in a pre-filled syringe, a glassampoule, or a plastic container.

The present disclosure also relates to a method of treating a diseaseusing the pharmaceutical composition or formulation according to thepresent disclosure.

The disease that can be treated using the pharmaceutical composition orformulation according to the present disclosure is not particularlylimited, and there is no limitation thereto, as long as it is a diseasethat can be treated with a drug used in combination with the PH20variant according to the present disclosure.

The disease that can be treated using the pharmaceutical composition orformulation according to the present disclosure may be cancer or anautoimmune disease, but is not limited thereto.

The cancer or carcinoma treatable with the pharmaceutical composition orformulation according to the present disclosure is not particularlylimited, and includes both solid cancers and blood cancers. Examples ofsuch cancers include skin cancer such as melanoma, liver cancer,hepatocellular carcinoma, gastric cancer, breast cancer, lung cancer,ovarian cancer, bronchial cancer, nasopharyngeal cancer, laryngealcancer, pancreatic cancer, bladder cancer, colorectal cancer, coloncancer, cervical cancer, brain cancer, prostate cancer, bone cancer,thyroid cancer, parathyroid cancer, kidney cancer, esophageal cancer,biliary tract cancer, testicular cancer, rectal cancer, head and neckcancer, cervical cancer, ureteral cancer, osteosarcoma, neuroblastoma,fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma,but is not limited thereto. Preferably, the cancer that can be treatedusing the pharmaceutical composition or formulation of the presentdisclosure may be selected from the group consisting of gastric cancer,colorectal cancer, breast cancer, lung cancer, and kidney cancer, but isnot limited thereto.

Autoimmune diseases treatable with the pharmaceutical composition orformulation according to the present disclosure include rheumatoidarthritis, asthma, psoriasis, multiple sclerosis, allergic rhinitis,Crohn's disease, ulcerative colitis, systemic erythematous lupus, type Idiabetes, inflammatory bowel disease (IBD), and atopic dermatitis, butis not limited thereto.

The present disclosure also provides a method of treating a diseaseincluding administering the pharmaceutical composition or formulationaccording to the present disclosure to a subject in need of treatment,and the present disclosure further provides the use of thepharmaceutical composition or formulation according to the presentdisclosure for the treatment of a disease.

Unless otherwise defined herein, the technical terms and scientificterms used in the present disclosure have meanings generally understoodby those of ordinary skill in the art. In addition, repeateddescriptions of the same technical configuration and operation as thoseof the related art will be omitted.

Hereinafter, the present disclosure will be described in further detailwith reference to the following examples. These examples are providedfor illustrative purposes only, and it will be obvious to those ofordinary skill in the art that these examples should not be construed aslimiting the scope of the present disclosure.

EXAMPLES Example 1. Formulation Development

Four trastuzumab subcutaneous injection formulations were prepared asshown in Table 6. Formulations 1 to 4 commonly contain 120 mg/mL oftrastuzumab and consist of 20 mM histidine/histidine-HCl (pH 5.5), 210mM trehalose, 10 mM methionine, and a PH20 variant. The difference amongformulations 1-4 is the concentration of a nonionic surfactant, whereinformulation 1: 0% polysorbate 20, formulation 2: 0.005% polysorbate 20,formulation 3: 0.04% polysorbate 20, and formulation 4: 0.1% polysorbate20.

TABLE 6 Composition of formulations Formulation Formulation FormulationFormulation 1 2 3 4 Antibody Trastuzumab (120 mg/mL) Buffer 20 mMhistidine/histidine-HCl Stabilizer 1 210 mM trehalose Stabilizer 2 10 mMmethionine Polysorbate 20 0% 0.005% 0.04% 0.1% Hyaluronidase HP46 of SEQID NO: 44 (2,000 units/mL)

Example 2. Measurement Using Spectrophotometer

Formulations 1 to 4 were left for 14 days at 45° C., and changes inprotein concentration were analyzed using a spectrophotometermanufactured by Beckman. Each sample was diluted with distilled water sothat the concentration of the sample was 0.4 mg/mL, and then absorbanceat 280 nm of the protein was measured using a spectrophotometer. In astability test under harsh conditions, i.e., at 45° C. for 14 days,there was no significant change in protein concentration of formulations1 to 4. However, the activity of hyaluronidase was rapidly reduced at45° C., and thus, in the present example, enzymatic activity was notmeasured (see FIG. 6).

Example 3. Investigation of Monomer Ratio of Trastuzumab in EachFormulation Using Size-Exclusion Chromatography

For size-exclusion chromatography analysis, an HPLC system availablefrom Shimadzu Prominence and a TSK-gel G3000SWXL (7.8×300 mm, 5 μm) anda TSK guard column (6.0×4.0 mm, 7 μm) were used. As a mobile phase, 0.2M potassium phosphate (pH 6.2) containing 0.25 M potassium chloride wasused. Analysis was performed for 35 minutes by applying an isocraticseparation mode at a flow rate of 0.5 mL/min. The sample was dilutedwith an analytical solvent so that the final concentration was 10 mg/mL,and after injecting 20 μL into the HPLC column, absorbance at 280 nm ofthe column eluate was recorded. The monomer ratio of trastuzumab in theHPLC chromatogram was calculated and graphed.

When size-exclusion chromatography analysis was performed in a stabilitytest under harsh conditions, i.e., at 45° C. for 14 days, formulations 1to 4 showed similar change patterns. The major changes were increases inhigh-molecular-weight (HMW) and low-molecular-weight (LMW) degradationproducts and a decrease in monomer content (about 1.5%), and there wasno significant difference according to formulation. In conclusion, as aresult of performing size-exclusion chromatography analysis in astability test under harsh conditions, i.e., at 45° C., there was nosignificant difference in stability profile between the formulationsaccording to the concentration of polysorbate 20 (0-0.1% (w/v)) (seeFIG. 1).

Example 4. Measurement of Protein Aggregation

Temperature of Formulations Containing Trastuzumab and HP46 Dynamiclight scattering (DLS) is used to analyze the denaturation properties ofproteins attributable to heat. In the present experiment, a change inthe size of a protein molecule according to the temperature change wasmeasured and used for the purpose of calculating the protein aggregationtemperature. For DLS analysis, a Zetasizer-nano-ZS instrument availablefrom Malvern, and a quartz cuvette (ZEN2112) were used. In the analysisprocess, the temperature was increased from 25° C. to 85° C. atintervals of 1° C., and the sample was diluted to 1 mg/mL using eachformulation buffer, and then 150 μL of the sample was added to thecuvette for analysis.

The aggregation temperature in formulation 1, not containing polysorbate20, was 74° C., and the aggregation temperature in formulations 2 to 4was 76° C. (see FIG. 2)).

Example 5. WCX Chromatography Measurements for Formulations ContainingTrastuzumab and HP46

For WCX chromatography analysis, a HPLC system available from ShimadzuProminence, and as columns, a TSKgel CM-STAT column (4.6×100 mm, 7 μm),a TSKgel guard gel CMSTAT (3.2 mm i.d.×1.5 cm), and the like were used.Mobile phase A is 10 mM sodium phosphate (pH 7.5) and mobile phase B is10 mM sodium phosphate (pH 7.2) containing 0.1 M NaCl. Analysis wascarried out for 55 minutes with a linear concentration gradient of 0-30%mobile phase B at a flow rate of 0.8 mL/min. The sample was diluted withmobile phase A so that the final concentration was 1.0 mg/mL, 80 μL ofthe sample was injected into HPLC, and then absorbance of a columneluate at 280 nm was recorded. The monomer ratio of trastuzumab in theHPLC chromatogram was calculated and graphed.

Formulations 1 to 4 showed similar change patterns when WCX analysis wasperformed in a stability test under harsh conditions, i.e., at 45° C.for 14 days. Specific changes include an increase in the relativecontent of acidic variants (approximately 30% change for 14 days), adecrease in the main peak relative content (approximately 44% change for14 days), and an increase in the relative content of basic variants(approximately 15% change for 14 days), and there was no significantdifference according to formulation. In conclusion, in the WCX analysisin a stability test under harsh conditions, i.e., at 45° C., proteinstability according to polysorbate 20 (0-0.1%) was similar (see FIG. 3).

Example 6. Formulation Development

Three types of trastuzumab subcutaneous injection formulations wereprepared as described in Table 7. Formulations 5 to 7 commonly include120 mg/mL of trastuzumab, 20 mM histidine/histidine-HCl (pH 5.5), 210 mMtrehalose, 10 mM methionine, and HP46. The difference among formulations5-7 is the ingredient of stabilizer 3: formulation 5: 0.04% polysorbate20, formulation 6: 50 mm Lys-Lys, and formulation 3: glycine.

TABLE 7 Composition of formulations Formulation Formulation Formulation5 6 7 Antibody Trastuzumab (120 mg/mL) Buffer 20 mMhistidine/histidine-HCl Stabilizer 1 210 mM trehalose Stabilizer 2 10 mMmethionine Stabilizer 3 0.04% 50 mM 50 mM polysorbate 20 Lys-Lys glycineHyaluronidase HP46 of SEQ ID NO: 44 (2,000 units/mL)

Example 7. Measurement Using Spectrophotometer

Formulations 5 to 7 were left for 14 days at 45° C., and changes inprotein concentration were analyzed using a spectrophotometermanufactured by Beckman. Each sample was diluted with distilled water sothat the concentration of the sample was 0.4 mg/mL, and then absorbanceof the protein at 280 nm was measured using a spectrophotometer. In astability test under harsh conditions, i.e., at 45° C. for 14 days,there was no significant change in protein concentration of formulations5 to 7. However, the activity of hyaluronidase was rapidly reduced at45° C., and thus, in the present example, enzymatic activity was notmeasured (see FIG. 6).

Example 8. Investigation of Monomer Ratio of Trastuzumab in EachFormulation Using Size-Exclusion Chromatography

For size-exclusion chromatography analysis, an HPLC system availablefrom Shimadzu Prominence and as columns, a TSK-gel G3000SWXL (7.8×300mm, 5 μm) and a TSK guard column (6.0×4.0 mm, 7 μm) were used. As amobile phase, 0.2 M potassium phosphate (pH 6.2) containing 0.25 Mpotassium chloride was used. An isocratic separation mode was applied ata flow rate of 0.5 mL/min for 35 minutes. The sample was diluted with ananalytical solvent so that the final concentration was 10 mg/mL, andafter injecting 20 μL of the sample into the HPLC column, absorbance at280 nm was measured. The monomer ratio of trastuzumab in the HPLCchromatogram was calculated and graphed.

When size-exclusion chromatography analysis was performed in a stabilitytest under harsh conditions, i.e., at 45° C. for 14 days, formulations 5to 7 showed similar change patterns. The major changes were increases inhigh-molecular-weight (HMW) and low-molecular-weight (LMW) impuritiesand a decrease in monomer content (about 1.5%), and there was nosignificant difference according to formulation. In conclusion, as aresult of performing size-exclusion chromatography analysis in astability test under harsh conditions, i.e., at 45° C., similar proteinstability was shown in 0.04% polysorbate 20, 50 mM Lys-Lys, and 50 mMglycine formulations (see FIG. 4).

Example 9. WCX Chromatography Analysis of Formulations ContainingTrastuzumab and HP46

For WCX chromatography analysis, a HPLC system available from ShimadzuProminence, and as columns, a TSKgel CM-STAT (4.6×100 mm, 7 μm), aTSKgel guard gel CMSTAT (3.2 mm i.d.×1.5 cm), and the like were used.Mobile phase A is 10 mM sodium phosphate (pH 7.5) and mobile phase B is10 mM sodium phosphate (pH 7.2) containing 0.1 M NaCl. Analysis wasperformed for 55 minutes by applying a separation mode of a linearconcentration gradient of 0-30% at a flow rate of 0.8 mL/min n. Thesample was diluted with mobile phase A so that the final concentrationwas 1.0 mg/mL, 80 μL of the sample was injected into HPLC, and thenabsorbance at 280 nm was recorded. The monomer ratio of trastuzumab inthe HPLC chromatogram was calculated and graphed.

Formulations 5 to 7 showed similar change patterns when WCX analysis wasperformed in a stability test under harsh conditions, i.e., at 45° C.for 14 days. Specific changes include an increase in the relativecontent of acidic variants (approximately 30% change for 14 days), adecrease in the main peak relative content (approximately 44% change for14 days), and an increase in the relative content of basic variants(approximately 15% change for 14 days), and there was no significantdifference according to formulation. In conclusion, as a result ofperforming WCX analysis in a stability test under harsh conditions,i.e., at 45° C., similar protein stability was shown in 0.04%polysorbate 20, 50 mM Lys-Lys, and 50 mM glycine formulations (see FIG.5).

Example 10. Stability Evaluation of HP46 According to Temperatures of40° C. and 45° C. in Subcutaneous Injection Formulations of Trastuzumaband HP46

To evaluate the stability of HP46 in subcutaneous injection formulationsof trastuzumab, trastuzumab (120 mg/mL) and PH20 (2000 units/mL) weremixed. At this time, the buffer used contained 20 mM Histidine (pH 5.5),210 mM trehalose, 10 mM methionine, and 0.04% polysorbate 20. Theenzymatic activity of a control sample was measured on day 0, and theexperimental samples were left at 40° C. or 45° C. for 1 day, and thenthe enzymatic activity of each sample was measured.

Each of a Herceptin subcutaneous injection formulation, trastuzumab+HW2,and trastuzumab+HP46 was left at 40° C. for 1 day, and then the activityof hyaluronidase was measured, and as a result, the respective casesexhibited activity of 51%, 47%, and 94%, which indicates that HP46 hadthe greatest thermal stability at 40° C. (see FIG. 6). In addition, theHerceptin subcutaneous injection formulation, trastuzumab+HW2, andtrastuzumab+HP46 were left at 45° C. for 1 day, and then the activity ofhyaluronidase was measured, and as a result, the Herceptin subcutaneousinjection formulation and trastuzumab+HW2 had no enzymatic activity, butthe enzymatic activity of trastuzumab+HP46 remained (see FIG. 6).

Example 11. Formulation Development

Three trastuzumab subcutaneous injection formulations were prepared asshown in Table 8. Formulations 8 to 10 commonly contain 120 mg/mL oftrastuzumab, 20 mM histidine/histidine-HCl (pH 5.5), 210 mM trehalose,10 mM methionine, and a PH20 variant. The difference among formulations8-10 is the concentration of a nonionic surfactant, wherein formulation8: 0% polysorbate 20, formulation 9: 0.005% polysorbate 20, andformulation 10: 0.04% polysorbate 20.

TABLE 8 Composition of formulations Formulation Formulation Formulation8 9 10 Antibody Trastuzumab (120 mg/mL) Polysorbate 20 0% 0.005% 0.04%Buffer 20 mM histidine/histidine-HCl Stabilizer 1 210 mM trehaloseStabilizer 2 10 mM methionine pH 5.5 Hyaluronidase HP46 of SEQ ID NO: 44(2,000 units/mL)

Example 12. Measurement Using Spectrophotometer

Formulations 8 to 10 were left for 14 days at 40° C., and changes inprotein concentration were analyzed using a spectrophotometermanufactured by Beckman. Each sample was diluted with distilled water sothat the concentration of the sample was 0.4 mg/mL, and then absorbanceat 280 nm of the protein was measured using a spectrophotometer. In astability test under harsh conditions, i.e. at 40° C. for 14 days, therewas no significant change in protein concentration of formulations 8 to10.

Example 13. Investigation of Monomer Ratio of Trastuzumab in EachFormulation Using Size-Exclusion Chromatography

For size-exclusion chromatography analysis, an HPLC system availablefrom Shimadzu Prominence and as columns, a TSK-gel G3000SWXL (7.8×300mm, 5 μm) and a TSK guard column (6.0×4.0 mm, 7 μm) were used. As amobile phase, 0.2 M potassium phosphate (pH 6.2) containing 0.25 Mpotassium chloride was used. Analysis was performed for 35 minutes byapplying an isocratic separation mode at a flow rate of 0.5 mL/min. Thesample was diluted with an analytical solvent so that the finalconcentration was 10 mg/mL, and after injecting 20 μL of the sample intothe HPLC column, absorbance at 280 nm was measured. The monomer ratio oftrastuzumab in the HPLC chromatogram was calculated and graphed.

When size-exclusion chromatography analysis was performed in a stabilitytest under harsh conditions, i.e., at 40° C. for 14 days, formulations 8to 10 showed similar change patterns. The major changes were increasesin high-molecular-weight (HMW) and low-molecular-weight (LMW)degradation products and a decrease in monomer content (about less than1.0%), and there was no significant difference according to formulation.In conclusion, as a result of performing size-exclusion chromatographyanalysis in a stability test under harsh conditions, i.e., at 40° C.,there was no significant difference in stability profile between theformulations according to the concentration (0-0.04%) of polysorbate 20(see FIG. 7).

Example 14. Measurement of Protein Aggregation Temperature forFormulations Containing Trastuzumab and HP46

Dynamic light scattering (DLS) is used to analyze the denaturationproperties of proteins attributable to heat in the protein drug field.In the present experiment, a change in the size of a protein moleculeaccording to the temperature change was measured and used for thepurpose of calculating the protein aggregation temperature. For DLSanalysis, a Zetasizer-nano-ZS instrument available from Malvern, and aquartz cuvette (ZEN2112) were used. In the analysis process, thetemperature was increased from 25° C. to 85° C. at intervals of 1° C.,and the sample was diluted to 1 mg/mL using each formulation buffer, andthen 150 μL of the sample was added to the cuvette for analysis.

The aggregation temperature in formulation 8, not containing polysorbate20, was 78.3° C., formulation 9 exhibited an aggregation temperature of77.3° C., and formulation 10 exhibited an aggregation temperature of77.7° C. In Example 13, no change in monomer ratio of the protein wasshown despite not containing polysorbate 20, and as a result ofcomparing the case of not containing polysorbate 20 with the case ofcontaining polysorbate 20, it was confirmed that there was no differencein aggregation between proteins. These results indicate that a minimumamount of polysorbate 20 is not necessarily required for subcutaneousinjection formulations of trastuzumab (see FIG. 8).

Example 15. WCX Chromatography Analysis for Formulations ContainingTrastuzumab and HP46

For WCX chromatography analysis, a HPLC system available from ShimadzuProminence, and as columns, a TSKgel CM-STAT column (4.6×100 mm, 7 μm),a TSKgel guard gel CMSTAT (3.2 mm i.d.×1.5 cm), and the like were used.Mobile phase A is 10 mM sodium phosphate (pH 7.5) and mobile phase B is10 mM sodium phosphate (pH 7.2) containing 0.1 M NaCl. Analysis wascarried out for 55 minutes with a linear concentration gradient of 0-30%mobile phase B at a flow rate of 0.8 mL/min. The sample was diluted withmobile phase A so that the final concentration was 1.0 mg/mL, 80 μL ofthe sample was injected into HPLC, and then absorbance of a columneluate at 280 nm was recorded. The monomer ratio of trastuzumab in theHPLC chromatogram was calculated and graphed.

Formulations 8 to 10 showed similar change patterns when WCX analysiswas performed in a stability test under harsh conditions, i.e., at 40°C. for 14 days. Specific changes include an increase in the relativecontent of acidic variants (approximately 10% change for 14 days), adecrease in the main peak relative content (approximately 40% change for14 days), and an increase in the relative content of basic variants(approximately 300% change for 14 days), and there was no significantdifference according to formulation. In conclusion, in the WCX analysisin a stability test under harsh conditions, i.e., at 40° C., proteinstability according to polysorbate 20 (0-0.04%) was similar (see FIG.9).

Example 16. Measurement of Enzymatic Activity for FormulationsContaining Trastuzumab and HP46

Turbidimetric assay for measuring enzymatic activity is a method ofmeasuring, by absorbance, the degree to which an aggregate is formed bybinding of residual hyaluronic acid to acidified albumin (BSA), and whenhyaluronic acid is hydrolyzed by PH20, the extent of binding to albuminis reduced, resulting in reduced absorbance. BTH (Sigma) as astandardized product was diluted to 1 unit/mL, 2 units/mL, 5 units/mL,7.5 units/mL, 10 units/mL, 15 units/mL, 20 units/mL, 30 units/mL, 50units/mL, and 60 unit/mL and prepared in each tube. Purified PH20variant samples were diluted with enzyme diluent buffer (20 mM Tris.HCl,pH 7.0, 77 mM NaCl, 0.01% (w/v) bovine serum albumin) to 100×, 300×,600×, 1200×, and 2400× and prepared in each tube. In fresh tubes, thehyaluronidase solution, having a concentration of 3 mg/mL, was diluted10-fold to a concentration of 0.3 mg/mL so that the volume of each tubebecame 180 μL. 60 μL of the sample containing hyaluronidase was added tothe diluted hyaluronic acid solution and mixed therewith, and allowed toreact at 37° C. for 45 minutes. After the reaction was completed, 50 μLof the reacted enzyme and 250 μL of an acidic albumin solution wereadded to each well of a 96-well plate and shaken for 10 minutes, andthen absorbance at 600 nm was measured using a spectrophotometer.

As a result of performing activity analysis in a stability test underharsh conditions, that is, at 40° C. for 14 days, it was confirmed thatthe higher the concentration of polysorbate 20, the greater thereduction in activity over time (see FIG. 10).

Example 17. Formulation Development

Three trastuzumab subcutaneous injection formulations were prepared asshown in Table 9. Formulations 11 to 13 commonly contain 120 mg/mL oftrastuzumab, 20 mM histidine/histidine-HCl (pH 5.5), 210 mM trehalose,10 mM methionine, and a PH20 variant. The difference among formulations11-13 is the concentration of a nonionic surfactant, wherein formulation11: 0% polysorbate 80, formulation 12: 0.005% polysorbate 80, andformulation 13: 0.04% polysorbate 80.

TABLE 9 Composition of formulations Formulation Formulation Formulation11 12 13 Antibody Trastuzumab (120 mg/mL) Polysorbate 80 0% 0.005% 0.04%Buffer 20 mM histidine/histidine-HCl Stabilizer 1 210 mM trehaloseStabilizer 2 10 mM methionine pH 5.5 Hyaluronidase HP46 of SEQ ID NO: 44(2,000 units/mL)

When size-exclusion chromatography analysis was performed in a stabilitytest under harsh conditions, i.e. at 40° C. for 14 days, formulations 11to 13 showed similar change patterns. The major changes were increasesin high-molecular-weight (HMW) and low-molecular-weight (LMW)degradation products and a decrease in monomer content (about less than1.0%), and there was no significant difference according to formulation.In conclusion, as a result of performing size-exclusion chromatographyanalysis in a stability test under harsh conditions, i.e. at 40° C.,there was no significant difference in stability profile between theformulations according to the concentration (0-0.04%) of polysorbate 80(see FIG. 11).

Example 18. WCX Chromatography Analysis for Formulations ContainingTrastuzumab and HP46

For WCX chromatography analysis, a HPLC system available from ShimadzuProminence and as columns, a TSKgel CM-STAT column (4.6×100 mm, 7 μm), aTSKgel guard gel CMSTAT (3.2 mm i.d.×1.5 cm), and the like were used.Mobile phase A is 10 mM sodium phosphate (pH 7.5) and mobile phase B is10 mM sodium phosphate (pH 7.2) containing 0.1 M NaCl. Analysis wascarried out for 55 minutes with a linear concentration gradient of 0-30%mobile phase B at a flow rate of 0.8 mL/min. The sample was diluted withmobile phase A so that the final concentration was 1.0 mg/mL, 80 μL ofthe sample was injected into HPLC, and then absorbance of a columneluate at 280 nm was recorded. The monomer ratio of trastuzumab in theHPLC chromatogram was calculated and graphed.

Formulations 11 to 13 showed similar change patterns when WCX analysiswas performed in a stability test under harsh conditions, i.e. at 40° C.for 14 days. Specific changes include an increase in the relativecontent of acidic variants (approximately 10% change for 14 days), adecrease in the main peak relative content (approximately 40% change for14 days), and an increase in the relative content of basic variants(approximately 300% change for 14 days), and there was no significantdifference according to formulation. In conclusion, in the WCX analysisin a stability test under harsh conditions, i.e. at 40° C., proteinstability according to polysorbate 80 (0-0.04%) was similar (see FIG.12).

Example 19. Measurement of Enzymatic Activity for FormulationsContaining Trastuzumab and HP46

Turbidimetric assay for measuring enzymatic activity is a method ofmeasuring, by absorbance, the degree to which an aggregate is formed bybinding of residual hyaluronic acid to acidified albumin (BSA), and whenhyaluronic acid is hydrolyzed by PH20, the extent of binding to albuminis reduced, resulting in reduced absorbance. BTH (Sigma) as astandardized product was diluted to 1 unit/mL, 2 units/mL, 5 units/mL,7.5 units/mL, 10 units/mL, 15 units/mL, 20 units/mL, 30 units/mL, 50units/mL, and 60 unit/mL and prepared in each tube. Purified proteinsamples were diluted with enzyme diluent buffer (20 mM Tris.HCl, pH 7.0,77 mM NaCl, 0.01% (w/v) bovine serum albumin) to 100×, 300×, 600×,1200×, and 2400× and prepared in each tube. In fresh tubes, thehyaluronidase solution, having a concentration of 3 mg/mL, was diluted10-fold to a concentration of 0.3 mg/mL so that the volume of each tubebecame 180 μL. 60 μL of the sample containing hyaluronidase was added tothe diluted hyaluronic acid solution, mixed therewith, and allowed toreact at 37° C. for 45 minutes. After the reaction was completed, 50 μLof the reacted enzyme and 250 μL of an acidic albumin solution wereadded to each well of a 96-well plate and shaken for 10 minutes, andthen absorbance at 600 nm was measured using a spectrophotometer.

As a result of performing activity analysis in a stability test underharsh conditions, that is, at 40° C. for 14 days, it was confirmed thatthe higher the concentration of polysorbate 80, the greater thereduction in activity over time (see FIG. 13).

Example 20. Formulation Development

Three types of rituximab formulations were prepared as described inTable 10. Formulations 14 to 16 commonly include 120 mg/mL of rituximab,20 mM histidine/histidine-HCl (pH 5.5), 210 mM trehalose, 10 mMmethionine, and a PH20 variant. The difference among formulations 14-16is the concentration of a non-ionic surfactant: formulation 14: 0%polysorbate 80, formulation 15: 0.005% polysorbate 80, and formulation16: 0.06% polysorbate 80.

TABLE 10 Composition of formulations Formulation Formulation Formulation14 15 16 Rituximab 120 mg/mL (±10) PS 80 0% 0.005% 0.06% Buffer 20 mMhistidine/histidine-HCl Stabilizer 1 210 mM trehalose Stabilizer 2 10 mMmethionine pH 5.5 Hyaluronidase HP46 of SEQ ID NO: 44 (2,000 units/mL)

When size-exclusion chromatography analysis was performed in a stabilitytest under harsh conditions, i.e. at 40° C. for 7 days, formulations 14to 16 showed similar change patterns. The major changes were increasesin high-molecular-weight (HMW) and low-molecular-weight (LMW)degradation products and a decrease in monomer content (less than about1.0%), and there was no significant difference according to formulation.In conclusion, as a result of performing size-exclusion chromatographyanalysis in a stability test under harsh conditions, i.e. at 40° C.,there was no significant difference in stability profile between theformulations according to the concentration (0-0.06%) of polysorbate 80(see FIG. 14).

Example 21. Measurement of Enzymatic Activity for FormulationsContaining Rituximab and HP46

Turbidimetric assay for measuring enzymatic activity is a method ofmeasuring, by absorbance, the degree to which an aggregate is formed bybinding of residual hyaluronic acid to acidified albumin (BSA), and whenhyaluronic acid is hydrolyzed by PH20, the extent of binding to albuminis reduced, resulting in reduced absorbance. BTH (Sigma) as astandardized product was diluted to 1 unit/mL, 2 units/mL, 5 units/mL,7.5 units/mL, 10 units/mL, 15 units/mL, 20 units/mL, 30 units/mL, 50units/mL, and 60 unit/mL and prepared in each tube. Purified proteinsamples were diluted with enzyme diluent buffer (20 mM Tris.HCl, pH 7.0,77 mM NaCl, 0.01% (w/v) bovine serum albumin) to 100×, 300×, 600×,1200×, and 2400× and prepared in each tube. In fresh tubes, thehyaluronidase solution, having a concentration of 3 mg/mL, was diluted10-fold to a concentration of 0.3 mg/mL so that the volume of each tubebecame 180 μL. 60 μL of the sample containing hyaluronidase was added tothe diluted hyaluronic acid solution, mixed therewith, and allowed toreact at 37° C. for 45 minutes. After the reaction was completed, 50 μLof the reacted enzyme and 250 μL of an acidic albumin solution wereadded to each well of a 96-well plate and shaken for 10 minutes, andthen absorbance at 600 nm was measured using a spectrophotometer.

As a result of performing activity analysis in a stability test underharsh conditions, that is, at 40° C. for 7 days, it was confirmed thatthe higher the concentration of polysorbate 80, the greater thereduction in activity over time (see FIG. 15).

Example 22. Measurement of Enzymatic Activity in Formulations ofCommercially Available Products not Containing Polysorbate

Two types of commercially available rituximab formulations were preparedas described in Table 11. Formulation 17 is a commercially availablebuffer for subcutaneous injection formulations, and formulation 18 is acommercially available buffer for intravenous injection formulations.Formulations 17 and 18 contain a PH20 variant and rituximab at 120 mg/mLand 100 mg/mL, respectively, but do not contain polysorbate 80 unlikeformulations of commercially available products.

TABLE 11 Composition of formulations Formulation Formulation 17 18Rituximab 120 mg/mL 100 mg/mL Buffer 20 mM 25 mM Sodium citratehistidine/histidine-HCl Stabilizer 1 210 mM trehalose 145 mM NaClStabilizer 2 10 mM methionine 10 mM methionine pH 5.5 6.5 HyaluronidaseHP46 of SEQ ID NO: 44 (2,000 units/mL)

Turbidimetric assay for measuring enzymatic activity is a method ofmeasuring, by absorbance, the degree to which an aggregate is formed bybinding of residual hyaluronic acid to acidified albumin (BSA), and whenhyaluronic acid is hydrolyzed by PH20, the extent of binding to albuminis reduced, resulting in reduced absorbance. BTH (Sigma) as astandardized product was diluted to 1 unit/mL, 2 units/mL, 5 units/mL,7.5 units/mL, 10 units/mL, 15 units/mL, 20 units/mL, 30 units/mL, 50units/mL, and 60 unit/mL and prepared in each tube. Purified proteinsamples were diluted with enzyme diluent buffer (20 mM Tris.HCl, pH 7.0,77 mM NaCl, 0.01% (w/v) bovine serum albumin) to 100×, 300×, 600×,1200×, and 2400× and prepared in each tube. In fresh tubes, thehyaluronidase solution, having a concentration of 3 mg/mL, was diluted10-fold to a concentration of 0.3 mg/mL so that the volume of each tubebecame 180 μL. 60 μL of the sample containing hyaluronidase was added tothe diluted hyaluronic acid solution, mixed therewith, and allowed toreact at 37° C. for 45 minutes. After the reaction was completed, 50 μLof the reacted enzyme and 250 μL of an acidic albumin solution wereadded to each well of a 96-well plate and shaken for 10 minutes, andthen absorbance at 600 nm was measured using a spectrophotometer.

As a result of performing activity analysis in a stability test underharsh conditions, that is, at 40° C. for 6 days, it was confirmed thathigh activity was maintained even in the formulations not containingpolysorbate 80, and particularly, formulation 18 maintained highactivity (see FIG. 16).

Example 23: Formulation Development

Four types of pembrolizumab formulations were prepared as described inTable 12. Formulations 19, 20, and 21 commonly include 25 mg/mL ofpembrolizumab, 10 mM histidine (pH 5.5), 7% sucrose, 10 mM methionine,and a PH20 variant. The difference among formulations 19-21 is theconcentration of a non-ionic surfactant: formulation 19: 0% polysorbate80, formulation 20: 0.005% polysorbate 80, and formulation 21: 0.02%polysorbate 80. Formulation 22 contains 25 mg/mL of pembrolizumab andconsists of 10 mM histidine (pH 5.5), 210 mM trehalose, 10 mMmethionine, 0.02% polysorbate 80, and a PH20 variant.

TABLE 12 Composition of formulations Formulation Formulation FormulationFormulation 19 20 21 22 Antibody Pembrolizumab (25 mg/mL) Buffer 10 mMhistidine (pH 5.5) Stabilizer 1 7%    7%   7% 210 mM sucrose sucrosesucrose trehalose Stabilizer 2 10 mM 10 mM 10 mM 10 mM methioninemethionine methionine methionine Polysorbate 80 0% 0.005% 0.02% 0.02%Hyaluronidase HP46 of SEQ ID NO: 44 (2,000 units/mL)

Example 24. Measurement Using Spectrophotometer

Formulations 19, 20, 21, and 22 were left for 7 days at 40° C., andchanges in protein concentration were analyzed using a spectrophotometermanufactured by Beckman. Each sample was diluted with distilled water sothat the concentration of the sample was 0.4 mg/mL, and then absorbanceof the protein at 280 nm was measured using a spectrophotometer.

In a stability test under harsh conditions, i.e. at 40° C. for 7 days,there was no significant change in protein concentration of formulations19 to 22.

Example 25. Investigation of Monomer Ratio of Pembrolizumab in EachFormulation Using Size-Exclusion Chromatography

For size-exclusion chromatography analysis, an HPLC system availablefrom Shimadzu Prominence and as columns, a TSK-gel G3000SWXL (7.8×300mm, 5 μm) and a TSK guard column (6.0×4.0 mm, 7 μm) were used. As amobile phase, 0.2 M potassium phosphate (pH 6.2) containing 0.25 Mpotassium chloride was used. Analysis was performed for 35 minutes byapplying an isocratic separation mode at a flow rate of 0.5 mL/min. Thesample was diluted with an analytical solvent so that the finalconcentration was 10 mg/mL, and after injecting 20 μL of the sample intothe HPLC column, absorbance of the column eluate at 280 nm was measured.The monomer ratio of pembrolizumab in the HPLC chromatogram wascalculated and graphed.

When size-exclusion chromatography analysis was performed in a stabilitytest under harsh conditions, i.e., at 40° C. for 7 days, formulations19, 20, 21, and 22 showed similar change patterns. There was nosignificant difference according to formulation in the change patternsof high-molecular-weight (HMW) and low-molecular-weight (LMW)degradation products. In conclusion, as a result of performingsize-exclusion chromatography analysis in a stability test under harshconditions, i.e., at 40° C., formulations 19, 20, 21, and 22 did notshow any significant difference, and there was also no differenceaccording to the type of sugar (see FIG. 17). These results wereconsistent with those of the cases of trastuzumab and rituximabaccording to the previous examples.

Example 26. Measurement of Enzymatic Activity for FormulationsContaining Pembrolizumab and HP46

A turbidimetric assay for measuring enzymatic activity is a method ofmeasuring, by absorbance, the extent to which an aggregate is formed bybinding of residual hyaluronic acid to acidified albumin (BSA), and whenhyaluronic acid is hydrolyzed by PH20, the extent of binding to albuminis reduced, resulting in reduced absorbance. BTH (Sigma) as astandardized product was diluted to 1 unit/mL, 2 units/mL, 5 units/mL,7.5 units/mL, 10 units/mL, 15 units/mL, 20 units/mL, 30 units/mL, 50units/mL, and 60 unit/mL and prepared in each tube. Purified proteinsamples were diluted with enzyme diluent buffer (20 mM Tris.HCl, pH 7.0,77 mM NaCl, 0.01% (w/v) bovine serum albumin) to 100×, 300×, 600×,1200×, and 2400× and prepared in each tube. In fresh tubes, thehyaluronidase solution, having a concentration of 3 mg/mL, was diluted10-fold to a concentration of 0.3 mg/mL so that the volume of each tubebecame 180 μL. 60 μL of the sample containing the enzyme was added tothe diluted hyaluronic acid solution, mixed therewith, and allowed toreact at 37° C. for 45 minutes. After the reaction was completed, 50 μLof the reacted enzyme and 250 μL of an acidic albumin solution wereadded to each well of a 96-well plate and shaken for 10 minutes, andthen absorbance at 600 nm was measured using a spectrophotometer.

As a result of performing activity analysis in a stability test underharsh conditions, i.e., at 40° C. for 7 days, it was confirmed that asthe concentration of polysorbate 80 increased, the reduction in activityover time was somewhat large. It was also confirmed that, when the sameamount of polysorbate 80 was included, the reduction in activity wassmaller in a trehalose-containing formulation than in asucrose-containing formulation (see FIG. 18).

Example 27. pH-Activity Profiles of HP46 and Wild-Type HW2

For an experiment for confirming the pH-activity profiles of HP46 andwild-type HW2, a microturbidimetric assay method was used. A hyaluronicacid buffer for dissolving hyaluronic acid as a substrate and an enzymebuffer for diluting the enzyme were prepared for each pH.

A total of three 96-well plates were prepared for a reaction between theenzyme and the substrate and designated as A, B, and C, and anexperiment was carried out.

A hyaluronic acid buffer at a pH of 4.0, 4.5, or 5.0 was prepared using20 mM acetic acid and 70 mM NaCl, and a hyaluronic acid solution at a pHof 5.5, 6.0, 6.5, 7.0, or 8.0 was prepared using 20 mM sodium phosphateand 70 mM NaCl. 20 mg of hyaluronic acid was dissolved in 10 mL of eachof the prepared hyaluronic acid buffers to prepare a final hyaluronicacid substrate solution, which was then diluted with each hyaluronicacid buffer prepared according to pH to prepare 500 μL of the resultantsolution to a concentration of 0.1 mg/mL, 0.25 mg/mL, 0.45 mg/mL, or 0.7mg/mL, and 100 μL of each solution was dispensed into each well of the96-well plate designated as A. The hyaluronic acid buffers, diluted andprepared according to concentration, were used as calibration curves formeasuring the concentration of hyaluronic acid.

An enzyme buffer at a pH of 4.0, 4.5, or 5.0 was prepared using 20 mMacetic acid, 0.01% (w/v) BSA, and 70 mM NaCl, and an enzyme buffer at apH of 5.5, 6.0, 6.5, 7.0, or 8.0 was prepared using 20 mM sodiumphosphate, 0.01% (w/v) BSA, and 70 mM NaCl.

HP46 and wild-type HW2 enzymes were diluted with the enzyme bufferprepared according to pH to 10 units/mL, and 50 μL of the resultantsolution was dispensed into each well of the 96-well plate designated asB.

50 μL of the sample was transferred from each well of the 96-well platedesignated as A to each well of the 96-well plate designated as B,followed by allowing a reaction to occur in a 37° C. shaking incubatorfor 45 minutes. 15 minutes before the reaction was completed, 200 μL ofan acidic albumin solution was dispensed into each well of the 96-wellplate designated as C and prepared, and when the enzymatic substratereaction was completed, 40 μL of the sample was transferred from eachwell of the 96-well plate designated as B to each well of the 96-wellplate designated as C, followed by allowing a reaction to occur for 20minutes. After 20 minutes, absorbance at 600 nm was measured, and theamount of hyaluronic acid remaining after the enzymatic substratereaction was calculated, and the active profiles of the enzymesaccording to pH were completed (see FIG. 19).

Example 28. Test for Pharmacokinetics Using Herceptin SubcutaneousInjection Formulation and Trastuzumab and HP46 in Sprague-Dawley Rats

To examine whether a subcutaneous injection formulation of trastuzumaband HP46 exhibits the same pharmacokinetic properties as those of aHerceptin subcutaneous injection formulation, an experiment wasconducted using 9-week-old Sprague-Dawley rats. The dose of administeredHerceptin and trastuzumab was 18 mg/kg of rat body weight, the amount ofrHuPH20 included in the Herceptin subcutaneous injection formulation was100 U, and the amount of HP46 was also 100 U. In the pharmacokinetictest, trastuzumab and HP46 showed the same Area Under the Curve (AUC) asthat of the Herceptin subcutaneous injection formulation (see FIG. 20).

INDUSTRIAL APPLICABILITY

A pharmaceutical composition according to the present disclosure can beused for subcutaneous injection and is also very stable, and theactivity of PH20 variants along with a drug, preferably an antibody drugor the like, can be maintained for a long time. Thus, the pharmaceuticalcomposition can contribute to a reduction not only in the cost ofproducing subcutaneous injection formulations but also in medical costs,and is very effective in terms of convenience of patients.

Although the preferred embodiments of the present disclosure have beendisclosed for Illustrative purposes, those skilled in the art willappreciate that various modifications, additions and substitutions arepossible, without departing from the scope and spirit of the inventionas disclosed in the accompanying claims.

REFERENCES

-   Bookbinder, L. H., Hofer, A., Haller, M. F., Zepeda, M. L.,    Keller, G. A., Lim, J. E., Edgington, T. S., Shepard, H. M.,    Patton, J. S., and Frost, G. I. (2006). A recombinant human enzyme    for enhanced interstitial transport of therapeutics. J Control    Release 114, 230-241.-   Borders jr., C. L. and Raftery, A. (1968) Purification and Partial    Characterization of Testicular Hyaluronidase. J Biol Chem 243,    3756-3762-   Chao, K. L., Muthukumar, L., and Herzberg, 0. (2007). Structure of    human hyaluronidase-1, a hyaluronan hydrolyzing enzyme involved in    tumor growth and angiogenesis. Biochemistry 46, 6911-6920.-   Chen, K. J., Sabrina, S., El-Safory, N. S., Lee, G. C., and    Lee, C. K. (2016) Constitutive expression of recombinant human    hyaluronidase PH20 by Pichia pastoris. J Biosci Bioeng. 122, 673-678-   Frost, G. I. (2007). Recombinant human hyaluronidase (rHuPH20): an    enabling platform for subcutaneous drug and fluid administration.    Expert Opin Drug Deliv 4, 427-440-   Hofinger, E. S., Bernhardt, G., and Buschauer, A. (2007) Kinetics of    Hyal-1 and PH-20 hyaluronidases: comparison of minimal substrates    and analysis of the transglycosylation reaction. Glycobiology 17,    963-971-   Kreidieh, F. Y., Moukadem, H. A., and Saghir, N. S. E. (2016)    Overview, prevention and management of chemotherapy extravasation.    World J Clin Oncol 7, 87-97.-   Thomas, J. R., Yocum, R. C., Haller, M. F., and Flament J. (2009)    The INFUSE-Morphine IIB Study: Use of Recombinant Human    Hyaluronidase (rHuPH20) to Enhance the Absorption of Subcutaneous    Morphine in Healthy Volunteers. J Pain Symptom Manag 38, 673-682

1. A pharmaceutical composition comprising: (a) a drug; and (b) a PH20variant, wherein the PH20 variant comprises one or more amino acidresidue substitutions selected from the group consisting of S343E,M345T, K349E, L353A, L354I, N356E, and I361T, in wild-type PH20 having asequence of SEQ ID NO:
 1. 2. The pharmaceutical composition according toclaim 1, wherein the PH20 variant comprises one or more amino acidresidue substitutions selected from the group consisting of L354I andN356E.
 3. The pharmaceutical composition according to claim 1, whereinthe PH20 variant further comprises one or more amino acid residuesubstitutions in one or more regions selected from the group consistingof an alpha helix region and a region corresponding to a linker regionthereof of the wild-type PH20 of SEQ ID NO:
 1. 4. The pharmaceuticalcomposition according to claim 3, wherein the alpha helix region of thewild-type PH20 of SEQ ID NO: 1 is an alpha-helix 8 region (S347 toC381), and the linker region is a linker region (A333 to R346) betweenalpha-helix 7 and alpha-helix
 8. 5. The pharmaceutical compositionaccording to claim 4, wherein the alpha-helix region and the regioncorresponding to a linker region thereof is T341 to N363, T341 to I361,L342 to I361, S343 to I361, I344 to I361, M345 to I361, or M345 to N363of the wild-type PH20 of SEQ ID NO:
 1. 6. The pharmaceutical compositionaccording to claim 4, wherein one or more regions selected from thegroup consisting of the alpha-helix 8 region (S347 to C381) and thelinker region (A333 to R346) between alpha-helix 7 and alpha-helix 8 ofthe wild-type PH20 of SEQ ID NO: 1 are substituted with one or moreamino acid residues of the amino acid sequence of a corresponding regionof Hya11.
 7. The pharmaceutical composition according to claim 1,wherein the PH20 variant comprises amino acid residue substitution(s) ofL354I and/or N356E, and further comprises amino acid residuesubstitution(s) at one or more positions selected from the groupconsisting of T341, L342, S343, 1344, M345, S347, M348, K349, L352,L353, D355, E359, I361, and N363.
 8. The pharmaceutical compositionaccording to claim 7, wherein the PH20 variant comprises amino acidresidue substitution(s) of L354I and/or N356E, and further comprises oneor more amino acid residue substitutions selected from the groupconsisting of T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E,L352Q, L353A, D355K, E359D, I361T, and N363G.
 9. The pharmaceuticalcomposition according to claim 7, wherein the PH20 variant comprises anamino acid residue substitution of M345T, S347T, M348K, K349E, L352Q,L353A, L354I, D355K, N356E, E359D, and I361T.
 10. The pharmaceuticalcomposition according to claim 9, wherein the PH20 variant furthercomprises one or more amino acid residue substitutions selected from thegroup consisting of T341S, L342W, S343E, I344N, and N363G.
 11. Thepharmaceutical composition according to claim 10, wherein the PH20variant comprises any one amino acid residue substitution selected fromthe following groups: (a) T341S, L342W, S343E, I344N, M345T, S347T,M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (b)L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I,D355K, N356E, E359D, and I361T; (c) M345T, S347T, M348K, K349E, L352Q,L353A, L354I, D355K, N356E, E359D, and I361T; (d) M345T, S347T, M348K,K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, and N363G; (e)I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E,E359D, and I361T; and (f) S343E, I344N, M345T, S347T, M348K, K349E,L352Q, L353A, L354I, D355K, N356E, E359D, and I361T.
 12. Thepharmaceutical composition according to claim 1, wherein the PH20variant further comprises deletion of one or more amino acid residues inat least one of a C-terminus and an N-terminus.
 13. The pharmaceuticalcomposition according to claim 12, wherein in the PH20 variant, one ormore amino acid residues are deleted by cleavage before an amino acidresidue selected from the group consisting of M1 to P42 at theN-terminus.
 14. The pharmaceutical composition according to claim 13,wherein in the PH20 variant, one or more amino acid residues are deletedby cleavage before amino acid residue L36, N37, F38, R39, A40, P41, orP42 at the N-terminus.
 15. The pharmaceutical composition according toclaim 12, wherein in the PH20 variant, one or more amino acid residuesare deleted by cleavage after an amino acid residue selected from thegroup consisting of V455 to L509 at the C-terminus.
 16. Thepharmaceutical composition according to claim 15, wherein in the PH20variant, one or more amino acid residues are deleted by cleavage afteran amino acid residue selected from the group consisting of V455 to S490at the C-terminus.
 17. The pharmaceutical composition according to claim16, wherein in the PH20 variant, one or more amino acid residues aredeleted by cleavage after amino acid residue V455, C458, D461, C464,I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478,I480, Y482, A484, P486, T488, or S490 at the C-terminus.
 18. Thepharmaceutical composition according to claim 1, wherein the PH20variant further comprises, at the N-terminus, a signal peptide derivedfrom human hyaluronidase-1 (Hya11), a human growth hormone, or humanserum albumin.
 19. The pharmaceutical composition according to claim 1,wherein the PH20 variant has an amino acid sequence selected from theamino acid sequences of SEQ ID NO: 5 to SEQ ID NO:
 50. 20. Thepharmaceutical composition according to claim 19, wherein the PH20variant has a sequence of SEQ ID NO:
 44. 21. The pharmaceuticalcomposition according to claim 1, wherein the drug is a protein drug, anantibody, a small molecule, an aptamer, RNAi, an antisense, or acellular therapeutic agent.
 22. The pharmaceutical composition accordingto claim 21, wherein the drug is an antibody, a soluble receptor, or anFc fusion protein thereof.
 23. The pharmaceutical composition accordingto claim 22, wherein the antibody binds to one or more antigens selectedfrom the group consisting of 4-IBB, integrin, amyloid beta,angiopoietin, angiopoietin analog 3, B-cell-activating factor (BAFF),B7-H3, complement 5, CCR4, CD3, CD4, CD6, CD11a, CD19, CD20, CD22, CD30,CD33, CD38, CD52, CD62, CD79b, CD80, CGRP, Claudin-18, complement factorD, CTLA4, DLL3, EGF receptor, hemophilia factor, Fc receptor, FGF23,folate receptor, GD2, GM-CSF, HER2, HERS, interferon receptor,interferon gamma, IgE, IGF-1 receptor, interleukin 1, interleukin 2receptor, interleukin 4 receptor, interleukin 5, interleukin 5 receptor,interleukin 6, interleukin 6 receptor, interleukin 7, interleukin 12/23,interleukin 13, interleukin 17A, interleukin 17 receptor A, interleukin31 receptor, interleukin 36 receptor, LAG3, LFA3, NGF, PVSK9, PD-1,PD-L1, RANK-L, SLAMF7, tissue factor, TNF, VEGF, and vWF.
 24. Thepharmaceutical composition according to claim 22, wherein the antibodyis one or more selected from the group consisting of utomilumab,natalizumab, etrolizumab, vedolizumab, bimagrumab, bapineuzumab,crenezumab, solanezumab, aducanumab, gantenerumab, AMG 780, MEDI 3617,nesvacumab, vanucizumab, evinacumab, tabalumab, lanalumab, belimumab,omburtamab, ravulizumab, eculizumab, mogamulizumab, otelixizumab,teplizumab, muromonab, tebentafusp, blinatumoma, REGN1979, ibalizumab,zanolimumab, itolizumab, efalizumab, inebilizumab, tafasitamab,loncastuximab tesirine, ocrelizumab, ublituximab, obinutuzumab,ofatumumab, rituximab, tositumomab, ibritumomab tiuxetan, epratuzumab,inotuzumab ozogamicin, moxetumomab pasudotox, brentuximab vedotin,vadastuximab talirine, gemtuzumab ozogamicin, daratumumab, isatuximab,alemtuxumab, crizanlizumab, polatuzumab vedotin, galiximab, eptinezumab,fremanezumab, galcanezumab, erenumab, zolbetuximab, lampalizumab,tremelimumab, zalifrelimab, ipilimumab, rovalpituzumab tesirine,cetuximab, depatuxizumab, zalutumumab, necitumumab, panitumumab,emicizumab, nipocalimab, rozanolixizumab, burosumab, farletuzumab,mirvetuximab soravtansine, dinutuximab, naxitamab, otilimab,margetuximab, pertuzumab, trastuzumab, trastuzumab deruxtecan,trastuzumab emtansine, trastuzumab duocarmazine, patritumab,anifrolumab, emapalumab, ligelizumab, omalizumab, dalotuzumab,figitumumab, teprotumumab, gebokizumab, canakinumab, daclizumab,basiliximab, dupilumab, mepolizumab, reslizumab, benralizumab,clazakizumab, olokizumab, sirukumab, siltuximab, sarilumab,satralizumab, tocilizumab, REGN88, secukinumab, ustekinumab,briakinumab, lebrikizumab, tralokinumab, ixekizumab, bimekizumab,brodalumab, brazikumab, guselkumab, risankizumab, tildrakizumab,mirikizumab, nemolizumab, spesolimab, relatlimab, narsoplimab,fasinumab, tanezumab, alirocumab, evolocumab, bococizumab,lambrolizumab, balstilimab, camrelizumab, cemiplimab, dostarlimab,prolgolimab, sintilimab, spartalizumab, tislelizumab, pembrolizumab,nivolumab, atezolizumab, avelumab, envafolimab, durvalumab, bintrafuspalpha, denosumab, elotuzumab, concizumab, marstacimab, infliximab,adalimumab, golimumab, certolizumab pegol, ozoralizumab, brolucizumab,ranibizumab, bevacizumab, faricimab, ramucirumab, and caplacizumab. 25.The pharmaceutical composition according to claim 22, wherein thesoluble receptor or the soluble receptor included in the Fc fusionprotein of the soluble receptor is selected from the group consisting ofa TNF-α soluble receptor, a VEGF soluble receptor, CTLA-4, interleukin 1soluble receptor, and LFA3 soluble receptor.
 26. The pharmaceuticalcomposition according to claim 25, wherein the Fc fusion protein of thesoluble receptor is selected from the group consisting of etanercept,aflibercept, abatacept, belatacept, rilonacept, and alefacept.
 27. Thepharmaceutical composition according to claim 1, wherein furthercomprising one or more selected from the group consisting of a buffer, astabilizer, and a surfactant.
 28. The pharmaceutical compositionaccording to claim 27, wherein the buffer comprises one or more selectedfrom the group consisting of malate, formate, citrate, acetate,propionate, pyridine, piperazine, cacodylate, succinate,2-(N-morpholino)ethanesulfonic acid (MES), histidine, Tris, bis-Tris,phosphate, ethanolamine, carbonate, piperazine-N,N′-bis(2-ethanesulfonicacid) (PIPES), imidazole, BIS-TRIS propane,N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),3-(N-morpholino) propanesulfonic acid (MOPS), hydroxyethyl piperazineethane sulfonic acid (HEPES), pyrophosphate, and triethanolamine; thestabilizer comprises one or more selected from the group consisting ofcarbohydrates, sugars or hydrates thereof, sugar alcohols or hydratesthereof, and an amino acid; and the surfactant comprises one or morenonionic surfactants selected from the group consisting ofpolyoxyethylene-sorbitan fatty acid ester, polyethylene-polypropyleneglycol, polyoxyethylene-stearate, polyoxyethylene alkyl ethers, apolyoxyethylene-polyoxypropylene copolymer, and sodium dodecyl sulfate(SDS).
 29. The pharmaceutical composition according to claim 28, whereinthe carbohydrates, the sugars, or the sugar alcohols comprise one ormore selected from the group consisting of trehalose or hydratesthereof, sucrose, saccharin, glycerol, erythritol, threitol, xylitol,arabitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol,inositol, volemitol, isomalt, maltitol, polyglycitol, cyclodextrin,hydroxylpropyl cyclodextrin, and glucose, and the amino acid comprisesone or more selected from the group consisting of glutamine, glutamicacid, glycine, lysine, lysilysine, leucine, methionine, valine, serine,selenomethionine, citrulline, arginine, asparagine, aspartic acid,ornithine, isoleucine, taurine, theanine, threonine, tryptophan,tyrosine, phenylalanine, proline, pyrrolysine, histidine, and alanine.30. The pharmaceutical composition according to claim 27, wherein thepharmaceutical composition comprises a histidine buffer providing a pHof 5.5±2.0, trehalose, and methionine.
 31. The pharmaceuticalcomposition according to claim 27, wherein the pharmaceuticalcomposition comprises a histidine buffer providing a pH of 5.5±2.0,trehalose, methionine, and polysorbate.
 32. The pharmaceuticalcomposition according to claim 31, wherein the pharmaceuticalcomposition comprises a histidine buffer providing a pH of 5.5±2.0,10-400 mM α,α-trehalose, 1-50 mM methionine, and 0.0000001% (w/v) to0.5% (w/v) of polysorbate.
 33. An injection formulation for subcutaneousinjection, the injection formulation comprising the pharmaceuticalcomposition according to claim 1.